Transducing frequencies of phage P22 lysates prepared from Salmonella typhimurium exponential cultures in minimal and nutrient broth media were compared. The assumption is that cells grown in a minimal medium will have one replication fork per replication unit, but cells in nutrient broth will have multiple replication forks; therefore, the frequency of genetic markers near the origin of replication will be higher in the nutrient broth culture. Analysis of transduction showed a gradient of marker frequencies from the highest (the cysG-ilv region) to the lowest (purE-trpB region) in both clockwise and counter clockwise directions. This supports our previous observation that chromosome replication proceeds bidirectionally from the origin between cysG (109 min on S. typhimurium map) and ilv (122 min) to a terminus in purE-trpB region (20 to 53 min). Since this method avoids possible artifacts of other methods, the results are assumed to reflect the sequence of chromosome replication in exponentially growing cells. Evidence for the existence of multiple replication forks in nutrient broth-grown cells was supported by the following: (i) the marker frequency data fitted the assumption of multiple replication fork formation; (ii) residual deoxyribonucleic acid increase after inhibition of protein synthesis to complete a round of chromosome synthesis which was 44% in cells grown in a minimal medium and 82% in those in nutrient broth; (iii) segregation patterns of the (3)H-thymidine-labeled chromosome strands during subsequent growth in non-radioactive medium were studied by autoradiography, and the number of replication points per chromosome per cell was estimated as 5.6 for the nutrient broth culture and 2.5 for the minimal medium culture. These data support a model of symmetrical and bidirectional chromosome replication.