The antibiotic heliquinomycin, which inhibits cellular DNA replication at a half-maximal inhibitory concentration (IC50) of 1.4-4 μM, was found to inhibit the DNA helicase activity of the human minichromosome maintenance (MCM) 4/6/7 complex at an IC50 value of 2.4 μM. In contrast, 14 μM heliquinomycin did not inhibit significantly either the DNA helicase activity of the SV40 T antigen and Werner protein or the oligonucleotide displacement activity of human replication protein A. At IC50 values of 25 and 6.5 μM, heliquinomycin inhibited the RNA priming and DNA polymerization activities, respectively, of human DNA polymerase-α/primase. Thus, of the enzymes studied, the MCM4/6/7 complex was the most sensitive to heliquinomycin; this suggests that MCM helicase is one of the main targets of heliquinomycin in vivo. It was observed that heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent in the absence of single-stranded DNA. In contrast, heliquinomycin at an IC50 value of 5.2 μM inhibited the ATPase activity of the MCM4/6/7 complex in the presence of single-stranded DNA. This suggests that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single-stranded DNA.