We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.