The accumulation of both activity and protein of a methyltransferase (MTase) from Aspergillus parasiticus, which catalyzes conversion of sterigmatocystin to O-methylsterigmatocystin in the aflatoxin pathway, was detected in fungal mycelia slightly before the onset of aflatoxin biosynthesis in the same cultures. MTase protein was identified in mycelial postmicrosomal (soluble protein) fractions by electrophoresis and subsequent immunoblotting using antiserum raised against purified MTase protein; MTase activity was determined by measuring the rate of conversion of sterigmatocystin to O-methylsterigmatocystin in the presence of soluble protein fractions. Using the above technique, it was determined that MTase protein as well as MTase activity increased sharply in mycelia 30 to 45 h after inoculation, shortly after which, mycelial growth rate began to decline. During the subsequent time interval (45 to 70 h after inoculation), a sharp increase in aflatoxin levels was detected in the culture medium. Results obtained from an experiment in which cycloheximide was added to cultures at various times to inhibit protein synthesis and from an experiment in which mycelial proteins were radiolabelled to identify newly synthesized proteins indicated that accumulation of MTase activity and protein in late growth phase mycelia is due to de novo protein synthesis.