Addition of Channa punctatus GnRH (cGnRH, 2 μg/well) to suspended pituitary cell (6 10 4 cells/well) with 2 mM Ca 2 + caused about 14% increase in cellular Ca 2 + as compared to controls after 15 min. Ca 2 + remained at the elevated level for a further 45 min. A concurrent decrease of Ca 2 + in the medium from 2 to 1.25 mM/well (400 μl medium) was observed during the first 15 min of cGnRH incubation. Addition of verapamil (3 μM/well) significantly inhibited cellular Ca 2 + increase and medium Ca 2 + decrease (p < 0.05). A sharp rise of gonadotropin (GtH) release from the pituitary cells in response to cGnRH plus Ca 2 + occurred between 5 and 15 min. GtH remained steady for an additional 15 min and was followed by a significant increase at 45 min (p < 0.01). cGnRH stimulated GtH synthesis transiently. Cycloheximide (20 μg/well) blocked the increase of GtH release from 30 min onward but had no effect on stimulated GtH release at 15 min in response to cGnRH. Addition of 0.5 mM dibutyryl cAMP stimulated the release and synthesis of GtH from 45 min onward. cGnRH plus Ca 2 + resulted in a peak of cellular cAMP at 15 min, which followed by a sharp decline after 30 min. Baseline levels were reached after 60 min. cGnRH plus Ca 2 + lead to a significant increase in cellular calmodulin (CaM). Stimulation of phosphodiesterase (PDE) activity could only be detected at 30 min, but was increased significantly at 45 min. There is a clear correlation between cAMP level, Ca 2 + , CaM, and PDE activity suggesting that a rise in cellular Ca 2 + results in an increase in CaM, which in turn stimulates the PDE activity and this affects degradation of cAMP. cGnRH plus Ca 2 + stimulated cellular calmodulin-dependent protein kinase II (CaM-kinase II) activity as early as 5 min. Increases in protein kinase A (PKA) activity were only noticed in the later (60 min) part of incubation. These results indicate two distinct phases of events in murrel pituitary cells in response to cGnRH: 1) cGnRH causes a rapid influx of extracellular Ca 2 + with a concurrent rise in cellular CaM-kinase II activity, resulting in an acute GtH release from the stored GtH, and 2) a sustained release of GtH, involving cAMP and PKA.