The phosphorylation and dephosphorylation of proteins is known to play an integral part in the metabolic processes of a cell 111. Whilst the importance of protein kinases and phosphorylation in regulating luteal cell steroidogenesis is widely accepted [2], the involvement of phosphoprotein phosphatases (PP) in the regulation of luteal cell function remains unclear. Of the four types of serinehhreonine PP's (PPl, PP2A, PP2B and PP2C) known to be active in cellular regulation 131, types PPI and PP2A are suggested to be involved in the regulation of intracellular mechanisms of secretory cells 141. The objective of this study was to determine the activity of PP's in rat luteal cells, and to examine the effects of inhibiting these enzymes. Female Sprague-Dawley rats (24-28 days old) were superovulated by sequential injections of pregnant mare serum gonadotrophin (Folligon, Intervet, U.K. Ltd. 50 IU) and human chorionic gonadotrophin (Chorulon, Intervet, U.K. Ltd. 25 IU) [ S ] . Whole luteinized rat ovaries were collagenasedigested and purified on continuous Percoll density gradients (40%) as previously described [S]. PPI and PP2A activity was assayed by measuring the release of '*P from 3ZP-radiolabelled glycogen phosphorylase a, using a commercially available in vitro assay kit (Gibco BRL, U.K.). The effects of PP inhibition on the dephosphorylation of endogenous substrates were examined by measuring 32P transfer from Y~~P-ATP, in vitro, onto endogenous luteal cell proteins, separated by SDS-polyacrylamide gel electrophoresis and visual ised by autorad iograph y . A range of structurally dissimilar inhibitors of PPI and PP2A were effective inhibitors (P PPI in vitro [3,7,81, inhibited PP activity with a minimum effective dose of 10'M, over the concentration range 10~'"M to IO~'M. Cantharidin, which also has dissimilar sensitivity, effecting PP2A>PPI in vitro [9], showed a minimum effective dose of lO~"M, over the concentration range