Monkey cell lines that constitutively synthesize 38.6 kd lac repressor protein and bear stably integrated chloramphenicol acetyl transferase (CAT) genes linked to a lac operator-containing SV40 early promoter-enhancer were generated. When grown in medium containing isopropyl beta-D-thiogalactoside (IPTG), these cells acquired a CAT+ phenotype. In contrast, when grown in parallel in medium lacking IPTG, the cells remained CAT-. Maximum induction of CAT activity occurred after 4 days of IPTG exposure. Three days after removal of IPTG, induced cells had reverted to CAT-. Specific CAT activity increased up to 60-fold after induction, while background activity in uninduced cells was similar to or only slightly above that of parental, CAT- cells. CAT activity increased stepwise over a wide range of IPTG concentrations. Thus lac repressor-operator complexes can form on primate chromosomes and stringently block transcription from an adjoining promoter.