A genome map of Staphylococcus carnosus TM300, an important micro-organism in the food industry and long used as a starter culture, was constructed by pulsed-field gel electrophoresis of DNA fragments obtained after digestion with NotI, SfiI and ApaI. The size of the chromosome was estimated to be 2590 kb. The fragments were assembled into a physical map using a combination of complementary methods including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. Fifteen genes or gene clusters were positioned on the physical map by Southern hybridization analysis. The map provides a basis for further analysis of the S. carnosus chromosome.