The transcriptional organization of the Escherichia coli hemolysin genes (hlyCABD) encoded by pSF4000 was examined. The use of different hemolysin gene-specific radiolabeled probes in blots containing isolated in vivo RNA revealed 4.0-kilobase hlyCA and 8.0-kilobase hlyCABD transcripts. The treatment of cells with rifampin just before RNA isolation showed the half-lives of these mRNAs to be 10.2 and 4.4 min, respectively. The 5' ends of the hly transcripts were 462 and 464 nucleotides from the putative initiation codon of hlyC based on a primer extension method of RNA mapping. Deletion analysis of pSF4000 combined with quantification of the hemolysin structural protein HlyA by immunoblotting confirmed that major control of HlyA expression occurs within a 168-base-pair PstI fragment located 433 base pairs upstream of the start of hlyC. A second recombinant plasmid, pANN202-312, encoding an E. coli hemolysin of different origin expressed 6-fold less total HlyA and 50-fold less extracellular HlyA than pSF4000 in identical cell backgrounds. The pANN202-312 recombinant had a different hly promoter, with the hly mRNA beginning 264 nucleotides upstream from the start of hlyC. We showed by RNA blotting that cells harboring pANN202-312 compared with pSF4000 have similar steady-state levels of the hlyCA transcript but they lack a consistently detectable hlyCABD transcript. We propose that one reason for the disparate levels of extracellular hemolysin produced by hemolytic E. coli is dissimilar levels of mRNA encoding in part the transport genes hlyB and hlyD.