Homocitrate synthase activity (encoded by the lys1 gene) catalyzes the first step of the lysine and penicillin pathway and is highly sensitive to feedback regulation by L-lysine. The transcript levels of the lys1 gene and the homocitrate synthase activity are high during the growth phase and decrease during the antibiotic production phase, except in the high penicillin producer strain AS-P-99 which maintained high levels of homocitrate synthase activity in cultures at 96 h and 120 h. The lys1 gene was overexpressed in Penicillium chrysogenum using additional copies of lys1 with its own promoter or under the control of the pcbC promoter in either autonomously replicating or integrative vectors. Transformants containing 3 to 32 additional copies of the lys1 gene were selected. Some of these transformants, particularly TI-C4 (integrative) and TAR-L9 (with autonomously replicating plasmids) showed very high levels of lys1 transcript and, in the case of TAR-L9, high levels of homocitrate synthase activity in cultures of 120 h. However, these transformants did not show increased alpha-aminoadipate or lysine pools. A mutant P. chrysogenum L(-)G(-) disrupted in the lys2 gene (therefore lacking the lysine branch of the pathway) showed increased a-aminoadipate levels and produced higher levels of penicillin than non-disrupted control strains. Overexpression of the lys1 gene in the L-G- mutant resulted in high homocitrate synthase levels but no additional increase of the a-aminoadipate pool or penicillin production levels. These results suggest that after amplification of the homocitrate synthase levels there are other limiting steps in the common stem of the lysine and penicillin pathways.