The uptake of 1-14C-DL-alpha-aminoadipate in resting mycelium of Penicillium chrysogenum Wis 54-1255 and its metabolism during benzylpenicillin formation were studied. The pH optimum for uptake at 25 degrees C was 6.4. Over a range of concentrations from 0.01--1.0 mM, approximately 45% of 1-14C-DL-alpha-aminoadipate was taken up by carbon-starved mycelium. 14CO2 was formed at a low rate, and the total formed amounted to only 1--3% of the 1-14C-DL-alpha-aminoadipate supplied. The intracellular pool of alpha-aminoadipate appears to be expandable, depending on the concentration of alpha-aminoadipate in the medium. The rate of penicillin synthesis depended on the intracellular concentration of alpha-aminoadipate. Penicillin biosynthesis achieved half of the maximum rate at an intracellular concentration of 0.06 nmol alpha-aminoadipate/mg dry cell weight. This low concentration, the result of adding 0.01 mM DL-alpha-aminoadipate to the medium, was sufficient to reverse the inhibition of penicillin biosynthesis caused by 10 mM extracellular L-lysine. Aminoadipate appears to be recycled during penicillin formation. Labeled alpha-ketoadipate was formed from alpha-aminoadipate to the extent of about 25%.