The synthesis of human chorionic gonadotropin (alpha)- and (beta)-subunits and alkaline phosphatase has been examined in HeLa S(,3) cultures. A variety of compounds which inhibit DNA synthesis lead to the increased production of PAP and the glycoprotein hormone subunits in HeLa cells. In general, compounds which influenced deoxynucleotide metabolism were good inducers of these ectopic proteins, while agents which altered DNA structure by intercalation or covalent binding were poor inducers. The greatest induction was achieved with sodium butyrate. Although a direct correlation between protein induction and DNA synthesis inhibition has not been demonstrated for all inducers, the dose-response curves and kinetics of induction with several compounds showed that inhibition of DNA synthesis generally occurred prior to increased protein accumulation at concentrations similar to those causing protein induction. Thus, it may be possible that inhibition of DNA synthesis may be required but is not sufficient for protein induction;Induction of ectopic products was synergistic (or at least additive) when intermediate inducers were added in combination with butyrate, suggesting different modes of action for these compounds. The simultaneous addition to cultures of various paired combinations of these intermediate inducers resulted in no greater induction than that observed with either drug alone, indicating similar or competing mechanisms among them;Addition of RNA or protein synthesis inhibitors to cell culture medium containing sodium butyrate, aphidicolin, or phosphonoformic acid abruptly ceased the production of hCG-(alpha) subunit indicating RNA and protein synthesis were both required for induction of this protein;The parallel RIA dose-response curves indicate similar immunologic characteristics among the uninduced, induced, and urinary hCG-(alpha) subunits. Alkaline phosphatases in inducer-treated HeLa S(,3) cells resembled the term placental enzyme in their inhibition profile to several inhibitors and thermostability.