CI-920 is a structurally novel, phosphate-containing polyene lactone antitumor agent isolated from a previously undescribed subspecies of Streptomyces pulveraceus cultured from a Brazilian soil sample. CI-920 was active against murine leukemia P388, and highly active and curative against L1210 leukemia in vivo. CI-920 was less active or inactive against the murine solid tumors tested. Daily administration for five to nine days was more effective against L1210 leukemia than a single dose or doses every four days. Given three times daily for five days, CI-920 was more toxic and less active. CI-920 had similar activity intravenously and intraperitoneally. Oral administration was inactive and nontoxic. Subcutaneous treatment was less effective and more toxic. Structure-activity relationship studies showed that the phosphate group was essential for antitumor activity in vivo and in vitro. Hydrolyzing the lactone ring also resulted in loss of antitumor activity, as did acetylation of the 6-hydroxyl group. Hydroxylation at the 5-position of the lactone ring resulted in partial retention of antitumor activity, but in greater toxicity to mice. Removal of the 13-hydroxyl group resulted in retention of high antitumor activity with approximately three-fold improvement in dose-potency. CI-920 is not cytotoxic to prokaryotic cells. CI-920 causes inhibition of biosynthesis of RNA and DNA in intact L1210 cells. Protein synthesis is also inhibited at higher drug concentrations. The inhibition of nucleic acid synthesis is not an antimetabolite effect, since pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates are not depleted. CI-920 does not cause DNA strand breakage, as measured by alkaline elution, and is not mutagenic in the Ames test at concentrations up to 200 micrograms/ml. CI-920 does not cause direct inhibition of RNA polymerase or DNA polymerase in permeabilized cells. It is possible that CI-920 must be metabolically activated within the target cells; alternatively it may interact with a component of chromatin other than DNA or the polymerases. Flow cytometry studies showed that growth-inhibitory levels of CI-920 caused accumulation of cells in the G2+M region. Higher drug concentrations caused an S-phase block. CI-920 is an inhibitor and irreversible inactivator of reduced folate membrane transport, and appears to enter cells by this receptor. L1210 cells selected for resistance to CI-920 are cross-resistant to methotrexate, and deficient in reduced folate transport.(ABSTRACT TRUNCATED AT 400 WORDS)