The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.