SummaryPhospholipase A2(PLA2) activity was measured in the reproductive organs of adult male rats. Phosphatidylethanolamine and phosphatidylcholine labelled with14C linoleic (lino‐PE lino‐PC) and arachidonic acid (ara‐PE ara‐PC) at the 2‐position were used as substrates. Lino‐PE was hydrolysed most strongly by homogenates of the distal cauda epididymis but the testis vas deferens and caput and corpus epididymis also contained hydrolytic activity. Ara‐PC and lino‐PC were hydrolysed by homogenates of the cauda epididymis and testis. No hydrolysis of ara‐PE was detected using homogenates of reproductive tissues.Chromatofocusing of testis homogenate resulted in the appearance of two active forms of PLA2with different pl‐values (6.5 and 5.6) when lino‐PE was used as substrate. Maximum activities of both enzymes with 1 mM Ca2+were observed at pH 9.5. These isoenzymes have marked differences in response to Cu2+Nethylmaleimide and p‐bromophenacyl bromide (p‐BPB). Cu2+and Nethylmaleimide had almost no effect on PLA2activity with a pi value of 6.5 but inhibited the other isoenzyme strongly; the latter was almost more resistant to p‐bromophenacyl bromide. Both enzymes hydrolysed lino‐PE most strongly.Chromatofocusing of an homogenate of cauda epididymis also revealed two isoenzymes of PLA2with different pl‐values (6.0 and 5.0). The latter form was resistant to p‐bromophenacyl bromide but was more sensitive to Triton X‐100 and sodium deoxycholate than was other isoenzyme. The pH optimum of the isoenzyme with a pi value of 6.0 ranged from 6.25 to 8.75 whilst the other isoenzyme was most active at pH 8.0–8.75. Both isoenzymes hydrolysed lino‐PE most actively whilst lino‐PC and a