H-19B is a lambdoid phage that carries the genes (stx-I) encoding the two toxin subunits of a Shiga-like toxin; Escherichia coli lysogens of H-19B are converted to toxin producers. Based on the determination of a 17-kb region of the H-19B genome and functional studies, we have identified the early regulatory region and associated genes of H-19B, as well as the location of the late regulatory region and the toxin and lysis genes. A comparative analysis of the sequence of the H-19B genome reveals the presence of ORFs and genes found in analogous positions on the genomes of a number of other lambdoid phages. A cloned genomic fragment that confers immunity to an infecting H-19B phage contains an ORF of an analogous size and genomic location for a repressor gene, adjacent to a putative operator region. The lambda replication genes, O and P, are conserved in H-19B except for a 39-bp insert in the O gene creating two new O protein-binding sites in the origin of replication (ori), giving H-19B six binding sites as opposed to the four sites found in lambda. We identify ORFs and sequences involved in transcriptional regulation encoding N-like antitermination systems like those found in other lambdoid phages and nearly identical to sequences found in phage HK97. Our functional studies show that these sequences support antitermination even though they contain significant differences from those of other lambdoid phages. We also identify ORFs and sequences analogous to the Q-p'R late antiterminators-promoters found in other lambdoid phages. The Shiga-like stx-I genes are located directly downstream of the promoter, p'R, for the late genes, and upstream of the lysis genes.