In New Zealand, goats and deer can be infected with isolates of Mycobacterium paratuberculosis of either the cattle or the sheep type. For epidemiological purposes, it can be desirable to know which type is involved. While these two types can be distinguished by RFLP analysis using IS900 or by PCR amplification of IS1311 followed by restriction endonuclease analysis, a simple PCR test would be advantageous. We have identified a 0.3 kb DNA fragment from an isolate of the sheep type that contains a tandem copy of a 12 bp sequence. The same DNA fragment but without the tandem copy of 12 bp was found to be present in an isolate of the cattle type. One flanking sequence of the 0.3 kb fragment differs between the two types. A PCR test utilising these differences was constructed and has been applied to sheep (4 isolates) and cattle (3 isolates) types of M. paratuberculosis and also to M. avium (5 isolates). Primers specific for the cattle type gave a PCR product only for isolates of the cattle type and primers specific for the sheep type gave a PCR product only for isolates of the sheep type. Neither primer set gave a product for isolates of M. avium. Because of the nature of the DNA sequence differences between the sheep and cattle isolates it should be possible to construct a multiplex PCR that distinguishes clearly between cattle and sheep isolates in a single reaction. This test should be useful in any situation where it is important to distinguish quickly and cheaply between isolates of the cattle and sheep types.