The molecular weight of the active unit of phospholipase A2 (PA2) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA2 activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 degrees C prior to irradiation, the sperm exhibited PA2 activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA2 was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA2 activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at -20 degrees C for 48 hr followed by a heating period of 10 min at 60 degrees C. Long-term storage of spermatozoa at -20 degrees C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 degrees C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 degrees C or -20 degrees C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA2 when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA2 was found and was sensitive to changes in temperature.