Soluble protein fractions from Escherichia coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity. Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.