The addition of glucose to batch cultures of Penicillium chrysogenumNA AS-P-78 reduced the biosynthesis of penicillin. This regulatory effect was also observed in penicillin biosynthesis by nitrogen-limited resting cells when cultures were previously grown in high concentrations of glucose. The effect of glucose was concentration-dependent in the range of 28-140 mM. Incorporation of L-[U-¹⁴C]valine into penicillin in nitrogen-limited resting cultures was reduced by 70% when cells were grown on 140 mm glucose, as compared with that grown on lactose. It was not affected when the sugar was added to the resting cell system, in which penicillin biosynthesis took place without growth. Fructose, galactose and sucrose exerted the regulatory effect to the same extent as glucose (64 to 70%). Lactose did not exert suppression of penicillin biosynthesis. Penicillin-synthesizing activity in control cultures with lactose reached a peak at 24 hours of incubation and decreased slowly thereafter, as studied with resting cell cultures in which further protein synthesis was blocked with cycloheximide. Glucose repressed the formation of penicillin-synthesizing enzymes, but had no effect on the activity of these enzymes. These results suggest that glucose represses but does not inhibit penicillin biosynthesis.