We have identified the polypeptides encoded by the haemolysin export genes from a haemolytic determinant 2001 carried by pLG570. This was previously cloned from an E. coli strain, serotype 04 isolated from a human urinary tract infection. Subclones from the recombinant plasmid pLG570 carrying hlyD analysed in vitro and in minicells showed that this gene is transcribed from an independent promoter and encodes a 53 Kd polypeptide. In contrast, detectable levels of the gene products encoded by hlyB were only observed when transcription presumably emanated from a vector promoter. This gene was found to encode at least two polypeptides apparently expressed from alternative translational start sites within a single reading frame. In minicells the major product was a 66 Kd polypeptide whilst after expression in vitro the major product was a 46 Kd polypeptide. Transposon mutagenesis leading to the synthesis of the expected truncated polypeptides was used to confirm the identity of the hlyD and the two hlyB products. Preliminary results suggest that the majority of the 53 Kd polypeptide is located in the inner membrane when cell envelopes from minicells and maxicells were fractionated using sarkosyl, although residual amounts of the 53 Kd polypeptide were also found in the outer membrane.