We describe a method for isolating and determining differences in gene expression between related bacterial strains. The method is based upon differences in mRNA expression. To demonstrate this procedure, cDNA generated from total RNA of Listeria monocytogenes serotype 1/2a was hybridized to total RNA from a Tn916 mutant of serogroup 1/2a (M3) that was deficient in the production of listeriolysin O, the product of the hly gene. The single-stranded cDNA fragments remaining after hybridization represent the difference in expressed genes between the two strains. These subtraction products were used as hybridization probes to identify the corresponding hly gene in a Southern hybridization.