Both water activity (a,) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 'a and 25°C on both substrates. At 0.98 a, and 25°C, a single colony of A. alternata grown on wheat extract agar produced 807 jig of AOH, 603 jag of AME, and 169 jig of AE per ml in 30 days. However, production of all three mycotoxins at 0.95 a, was less than 40% of these amounts. Little toxin was produced at 0.90 a,. Changing temperature and a, altered the relative amounts of the different toxins produced on agar. At 15°C and 0.98 a,, maxima of 52 jag of AOH and 25 jxg of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 ,ug/ml after 40 days. At 15°C and 0.95 aw, production was, respectively, 62, 10, and 5 ,ug/ml after 40 days. All three metabolites were produced at 5°C and 0.98 to 0.95 a, and at 30°C and 0.98 to 0.90 a,. On wheat grain at 25°C and 0.98 to 0.95 aw, more AME was produced than AOH or AE, but at 15°C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25°C and 0.90 aw, but production of AME was inhibited at 30°C and 0.95 a, or less.