Trefoil proteins contain a conserved domain of distinctive structure. Three human trefoil proteins have been described to date of which the human spasmolytic polypeptide (hSP) and pNR-2/pS2 proteins have a similar pattern of expression in normal tissues. The genes encoding these two proteins were isolated from a human DNA library. Preliminary experiments suggested that some recombinants contained both genes. Southern hybridisation showed that all the recombinants were derived from a single stretch of DNA spanning 45 kb and suggested that the hSP gene was located downstream of the pNR-2/pS2 gene. Further experiments demonstrated that the two genes are transcribed in the same direction and that the distance between the 3' end of the pNR-2/pS2 gene and the 5' end of the hSP gene is 12.5 kb. The close linkage of these two genes is evidence that they have evolved by gene duplication and that their similar pattern of expression in normal tissues could result from the retention of common regulatory elements.