1 9 F-MRS (magnetic resonance spectroscopy) was used to study the pharmacokinetics of 5-fluorouracil (5-FU) in human (HT29) tumour xenografts, with and without pretreatment of the mice using either thymidine (40min) or interferon-α (2 and 24h). A 200mg/kg i.p. bolus dose of 5-FU was eliminated from control tumours with a t 1 / 2 of 25.4+/-2min (mean+/-SEM, n=11), while both thymidine (500mg/kg) and interferon (50000IU/mouse) significantly increased t 1 / 2 to 36.5+/-6.1 (n=5) and 48.1+/-13.6min (n=4), respectively (P=0.04, Gabriel's ANOVA). Thymidine increased 5-FU anabolism to cytotoxic 5-fluoronucleotides, and decreased the amount of tumour catabolites; the latter probably recirculated from liver since isolated HT29 cells did not catabolise 5-FU. These in vivo observations were confirmed by 1 9 F-MRS quantification of tumour extracts. Interferon did not significantly affect 5-FU metabolism in the tumour or liver, nor the 5-FU t 1 / 2 in liver. Treatment of tumours with 5-FU or interferon had no effect on tumour growth, whereas the combination strongly inhibited growth. 3 1 P-MRS of HT29 tumours showed that 2 and 24h after i.p. injections of interferon there was a significant increase in the pH i n t of 0.3+/-0.04 units (P=0.002), while pH e x t and the tumour NTP/Pi ratio were unchanged. The large increase in the negative pH gradient (-ΔpH) across the tumour plasma membrane caused by interferon suggests the ΔpH may be a factor in tumour retention of 5-FU, as recently shown in isolated tumour cells.