Twenty genes encoding enterotoxin and enterotoxin-like proteins have been described in Staphylococcus aureus strains. Five of these occur commonly in the enterotoxin gene cluster (egc: selo, selm, sei, seln and seg). In the sei-seln intergenic region, two pseudogenes, yent1 and yent2, can be present or an additional gene designated selu or a variant selu v . Whilst frequencies of loci bearing pseudogenes (egc1) or the selu gene (egc2) have been reported, the distinction between selu-bearing and selu v -bearing (egc3) loci has rarely been made. A PCR-RFLP procedure involving cleavage of the sei-seln intergenic region by restriction endonuclease BbvI or TseI was developed that allowed differentiation of selu + and selu v + loci. In addition, PCR primers were designed to yield a 203 bp amplimer for sequencing of a selu or selu v intragenic region, which encompassed ten signature nucleotide differences. A total of 43 egc + human nasal isolates and 53 egc + bovine, ovine, caprine, leporine and gallinaceous isolates were egc typed and agr typed. None of the animal isolates was of agr type III. A total of 12 out of 17 egc3 + human nasal isolates were of agr type III, the other 5 being agr type I. On the basis of representative multilocus sequence typing, agr type III/egc3 + strains belonged to CC30. Human nasal isolates bearing an egc1 locus were distributed evenly across agr types I, II and III. Only two nasal isolates had an egc2 locus. All 14 agr type IV isolates, only 1 of which was of human origin, possessed an egc2 locus. The agr IV nasal isolate was fusidic acid sensitive and was found to be ST123 (CC121). There were strong associations between bovine, leporine and gallinaceous S. aureus clonal types and egc locus types. The PCR-RFLP procedure was used to screen an additional 45 S. aureus isolates from dogs, cats, rats, pigs and horses for egc locus types. Of these, 33 were egc " . Six equine isolates were selu + . One canine and three porcine isolates possessed pseudogenes yent1 and yent2. One porcine and one canine isolate each had the selu v gene. Putative relationships between disease-causing propensity and egc type need (re-)evaluation.