The mechanism of action of a novel anti-HIV antibiotic, pradimicin A, has been studied using cell-free (MT-4 cells) and cell to cell (MOLT-4 and MOLT-4/HIVHTLV-3B cells) HIV infection systems. The data indicate that (1) preincubation of the cells with HIV at 0 degrees for 1 hr, followed by the addition of pradimicin A and further incubation at 37 degrees, resulted in a complete inhibition of infection while preincubation at 37 degrees did not. Similar data were obtained with the coculture between MOLT-4 and MOLT-4/HIV cells. (2) Virus treated with pradimicin A followed by washing did not show any inhibition on subsequent HIV binding to cells. (3) The inhibitory effect of pradimicin A on HIV infection was prevented by mannan but not by other sugars tested. Mannan, however, did not interfere with the anti-HIV activity of other known inhibitors. (4) Use of EGTA suggested that pradimicin A required Ca ions to exert its anti-HIV activity. These data imply that pradimicin A inhibits an early step in HIV infection, probably through its binding to mannose residues of HIV glycoprotein.