We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. These Kmr insertions were then crossed directly into the bacterial chromosome. We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants. We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes. The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT. We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-1 and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively. Of these genes, only the functions of usg-2 (pdxB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-1 or dsg-1 for function, usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37 degrees C. In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter. The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT. The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons.