The effect of lincomycin on the production of TEM β-lactamase by E. coli was studied to elucidate the mechanism of the stimulation by this antibiotic of the production of enterotoxins by certain bacteria. TEM β-lactamase was consistently measurable by a microiodometric assay. Lincomycin enhanced the activity of the enzyme encoded on the plasmid pBR322 2.4 times, while chromosomal β-galactosidase activity was inhibited. Lincomycin also stimulated the production of β-lactamase coded for by the bla gene integrated into the chromosome, but the antibiotic did not stimulate that of chloramphenicol acetyltransferase encoded on a ColEl-derived plasmid. The effect was independent of the copy number of the gene, suggesting that the effect takes place after transcription.The amount of bla mRNA was measured by [(3)H]-uridine pulse-labeling of mRNA followed by DNA-RNA hybridization. Lincomycin caused a 1.8 to 2.4-fold increase in the amount of bla mRNA. The possible presence of the repressor of the bla gene was examined by transducing the excess bla promoter region into E. coli 112-2, which carries Tn3 on its chromosome, but the repressor was not detected. The effect of bla promoters was examined by the construction of promoter deletion mutants of pBR322, each of which lacked one of two bla promoters. Lincomycin stimulated the production of β-lactamase coded by these plasmids at the same level as that coded by original pBR322, indicating that the effect was not due to increased transcription initiation. Preliminary examination of the degradation rate of bla transcripts showed that lincomycin increased half-life of bla mRNA.