Rapid and reliable in vitro detection methods are lacking for pathogenic leptospires such as Leptospira interrogans. This study investigated the use of luminescence to replace existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with a theoretical detection limit of less than 10(4) leptospires. A comparison of enumeration by a standard method (counting by dark field microscopy) and luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R(2)=0.766), though variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays including an MIC determination, extracellular matrix binding experiment, and complement killing experiment were conducted. In each case the results obtained with luminescence matched those obtained by traditional means with high correlations (binding assay R(2)=0.916, complement killing assay R(2)=0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of growth phase or particular assay conditions, luminescence was found to be a quick, reliable and highly sensitive in vitro detection method for leptospires that has the potential to replace more time consuming methods of enumeration.