Lincomycin increased the TEM-2 beta-lactamase activity of Escherichia coli K-12 cells carrying plasmid RP4 at a concentration which slightly inhibited cell growth. In a control culture beta-lactamase activity reached its maximal level in late log phase, whereas when lincomycin was present beta-lactamase activity continued to increase into the stationary phase. Lincomycin (100 micrograms/ml) inhibited both cell growth and protein synthesis by about 35% but stimulated beta-lactamase activity 2.5-fold per ml of culture and about 4-fold per cell after 20 h of growth. The amount of beta-lactamase produced in each culture was also compared by densitophotometry of a stained sodium dodecyl sulfate-polyacrylamide gel. The relative values were in good agreement with the relative enzyme activities, indicating that the stimulatory effect of lincomycin was due to an increase in the amount of beta-lactamase protein. Inactivation of beta-lactamase appeared to be faster when lincomycin was present. This was determined by measuring the decrease in beta-lactamase activity when phenethyl alcohol was present to prevent maturation of the enzyme. There was no significant difference in plasmid copy number between the cells grown in the presence or absence of lincomycin. These results indicate that lincomycin stimulates transcription, translation, or translocation of beta-lactamase.