Most media used for in vitro fertilization (IVF) in mammals contain bicarbonate (HCO - 3 ) and it is reported to be essential for IVF of pig oocytes (Suzuki et al., Reprod. Fertil. Dev. 6:221-227, 1994). However, according to Berger and Horton (Gamete Res. 19:101-111, 1988), boar spermatozoa can penetrate zona-free hamster oocytes in a Trisbuffered medium (TBM) which does not contain HCO - 3 . Therefore, it is hypothesized that HCO - 3 is not essential during IVF of pig oocytes. In experiment 1, cumulus oocyte complexes were cultured in NCSU 23 medium containing 10% porcine follicular fluid, 0.1 mg/ml cysteine and with 10 IU/ml eCG, 10 IU/ml hCG for 22 h, and then oocytes were cultured without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed boar spermatozoa for 6 h in a modified TBM without antibiotics (mTBM) containing 5 mM caffeine with 0.1 or 0.4% BSA. In experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0-5 mM). In experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca + 2 (0.5-10 mM). Data from three replicates were analyzed by ANOVA and fisher's protected LSD test. Supplementation of mTBM with 0.1 or 0.4% BSA resulted in a high penetration rate (98.9±1.1 and 95.4±2.3, respectively) and a high rate of polyspermy (93.3±3.3 and 96.4±2.0, respectively). However, the mean number of sperm (MNS) per oocyte was significantly (P<0.001) higher at 0.4% compared with 0.1% BSA (7.2±0.2 vs 3.8±0.4). In the absence of caffeine, penetration rate, polyspermy and MNS per oocyte were 47.7±2.0%, 33.9±1.6% and 1.5±0.1, respectively. The presence of 1-5 mM caffeine significantly (P<0.001) increased the penetration rate (94.5±1.4-96.6±1.8%) with no differences among different caffeine concentrations. Polyspermy (87.5±1.8, 86.3±8.3 and 95.2±2.5% at 1, 2.5 and 5 mM, respectively) and MNS per oocyte (3.4±0.3, 4.3±0.2 and 4.6±0.3 at 1, 2.5 and 5 mM, respectively) increased significantly (P<0.05) with increasing caffeine concentrations, although no difference was observed between 2.5 and 5 mM. In exp. 3, no penetration was observed in the presence of 0.5 mM Ca + 2 . Penetration rate increased significantly (P<0.001) with increasing Ca + 2 concentration (12.2±0.7, 68.8±3.6, 87.3±1.7 and 92.4±2.9% at 2.5, 5. 7.5 and 10 mM, respectively) although no difference was observed between 7.5 and 10 mM. All the oocytes penetrated at 2.5 mM were monospermic. A significant (P<0.001) increase in polyspermic penetration was observed at 5 (29.4±2.4%), 7.5 (62.5±3.6%) and 10 mM (84.4±5.2%). MNS per oocyte did not differ between 2.5 (1.0+0.0) and 5 mM (1.4+0.0), but increased significantly (P<0.001) at 7.5 (2.6+0.3) and 10 mM (3.5+0.2) Ca + 2 . The results clearly indicate that HCO - 3 is not necessary in the fertilization medium for the boar spermatozoa to penetrate pig oocytes. Furthermore, Ca + 2 seems to play a major role in modulating the ability of spermatozoa to penetrate into oocytes possibly by stimulating capacitation and/or acrosome reaction.