The mechanism of action of geldanamycin, a benzoquinoid ansamycin, was investigated with murine lymphoblastoma L5178Y cells. The agent inhibited the cell growth at concentrations over 0.01 micrograms/ml. The antibiotic blocked DNA synthesis more markedly than RNA and protein syntheses. Mitosis was not significantly affected by the drug in the cells synchronized with demecolcine (Colcemid). The antibiotic did not interfere with in vitro assembly of tubulin. In the synchronized cells, strong inhibition of DNA synthesis was observed when geldanamycin was introduced into the culture prior to S phase of the cell cycle. The degree of inhibition was stronger with prolongation of incubation period and with increase of DNA synthesis rate. The results suggested that initiation of DNA synthesis or S phase is affected by the drug. DNA degradation was not significantly induced in vivo by the antibiotic. Geldanamycin blocked DNA polymerase alpha more markedly than beta and gamma. The degree of inhibition depended up on concentrations of enzyme but not upon those of template, suggesting a drug-enzyme interaction. IC50 for DNA polymerase alpha was 10 micrograms/ml and for DNA polymerase beta 100 micrograms/ml at low concentrations of enzyme. The inhibition of DNA polymerase alpha by the antibiotic was non-competitive and Ki was 20 microM.