Three strains of Penicillium chrysogenum (Q176, D6/1014 A, and P2), which have different rates of penicillin biosynthesis, were examined with respect to glutamate pools as well as activities of NADP- and NAD-glutamate dehydrogenase and regulation of the NADP-specific enzyme. Penicillin biosynthesis was accompanied by an increase in the internal glutamate pool in all P. chrysogenum strains, which was more pronounced in higher-producing strains. An increase in the glutamate to glutamine ratio was also noted. No striking differences in the glutamate pools were observed in the three strains under growing, non-penicillin-producing conditions. NADP-glutamate dehydrogenase (NADP-GDH) activity was higher in higher-producer organisms under nonproducing conditions; Under conditions allowing penicillin production, a general correlation between the relative penicillin productivity of strains and the relative NADP-GDH specific activity in those strains existed until late in the fermentation. The most productive strain (P2) showed a significant increase in NADP-GDH specific activity late in the fermentation (80–120 h), and the strain of intermediate penicillin productivity (D6/1014 A) exhibited lower and relatively constant NADP-GDH specific activity throughout the fermentation. However, the least productive strain (Q 176) exhibited a biphasic behavior in NADP-GDH specific activity: at 40 and 50 h NADP-GDH specific activity was similar to, but slightly lower than, that of the intermediate strain, and at 80 h it had fallen to a very low activity, but the NADP-GDH specific activity of Q 176 then rebounded at 120 h to a level exceeding the activity of the most productive strain. Q 176 was the only strain that showed significant NAD-GDH specific activity. Significant but decreasing NADP-GDH specific activities were obtained under non-penicillin-producing conditions. None of the strains tested exhibited significant NAD-GDH specific activities under nonproducing conditions. NADP-GDH was purified from the three strains by modification of published procedures involving Procion Red HE-3B and Mono Q ion-exchange chromatography. The enzyme in all strains exhibited similar kinetic, physical, and regulatory properties except for the kinetics with α-ketoglutarate, where strains D6/1014 A and P2 exhibited higher affinity and cooperativity.