A derivative of Tn917 was constructed, referred to as Tn917-lac, which is capable of generating fusions that connect the transcripts of Bacillus subtilis chromosomal genes to the coding sequence of the lacZ gene of Escherichia coli. Two independent insertions of Tn917-lac into the gltA gene and one insertion into the trpE gene (in the trpEDCFBA operon) of B. subtilis were studied in detail, and the results confirmed that Tn917-lac-mediated transcriptional fusions produce levels of beta-galactosidase that reflect accurately the regulated expression of interrupted genes. To facilitate these studies, a procedure was developed that permits the analysis of Tn917-lac-mediated fusions in partial diploids where insertional mutations are complemented by an intact copy of the interrupted genes. Tn917 is known to function efficiently in bacteria representing three quite different Gram-positive genera (Streptococcus, Bacillus, and Staphylococcus) and is known to display a relatively high degree of randomness in its insertions into bacterial genomes, making it likely that Tn917-lac will be useful for the identification and study of many kinds of regulated genes in a wide range of Gram-positive species.