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Cell-free extracts of fungal mycelia of two non-aflatoxigenic isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were examined for enzyme activities involved in the latter stages of aflatoxin biosynthesis. Post-microsomal fractions (105 Kxg supernatant) of both SRRC 163 (ATTC 56774) and SRRC 2043 were able to convert sterigmatocystin (ST) to O-methylsterigmatocystin (OMST); whereas the microsomal (105 Kxg pellet) preparation of only SRRC 163 was able to convert OMST to aflatoxin B1 (AFB1). A mixture of the microsomal fraction of SRRC 163 and post-microsomal fraction of SRRC 2043 converted ST to AFB1. Electrophoretic protein separations of the microsomal fractions of the two A. parasiticus isolates on non-denaturing or denaturing polyacrylamide gel electrophoresis (PAGE) provided equivalent protein profiles.