Two-dimensional zymography which combines two-dimensional electrophoresis with zymography was used to analyze proteases and other proteins produced by different phase variants of two strains of Photorhabdus luminescens. Both the primary and secondary phases of P. luminescens strains Hp and Hm secreted proteases. The protease in P. luminescens Hp has a molecular weight (Mr) of 57,000 and an isoelectric point (pI) of 4.4 whereas that in P. luminescens Hm has an Mr of 59,000 and pI of 4.9. Several putative protease degradation products were clearly visible in the zymograms from both bacterial strains. Two-dimensional zymography also showed that several secretory proteins were present only in particular phase variants and therefore could be used as specific markers. Unexpectedly, the two-dimensional zymography revealed that a nonsecretory protease with an Mr of 47,000 and a pI of 4.0 was present in the cell extracts of all phases of both P. luminescens Hp and Hm. The application of the two-dimensional zymography for the identification of other enzymes was also discussed.