The two-locus gametophytic incompatibility system in perennial ryegrass (Lolium perenne L.) is not always fully effective: obligate selfing of plants sieves self-compatible pollen mutants, and self-fertility becomes fixed in subsequent generations. Self-compatibility (SC) was investigated in an F2 family. In vitro self-pollinations were analysed and recorded and plants were classified as being either partially or fully compatible. Distorted segregation ratios of markers on linkage group (LG) 5 were found, which indicate the possible presence of a gametophytic SC locus. Interval linkage analysis of pollen compatibility after selfing confirmed that this distortion was due to a locus (T) analogous to the S5 locus of rye. However, even though markers in this region were, on average, less than 1 cM apart, the minimum number of plants possessing the unfavoured allele was never less than 6% for any marker locus. We proved that this was because of the presence of another SC locus, exhibiting gametophytic selection, segregating in this population and identified by interval mapping analysis of compatibility classes of in vitro self-pollinations. This locus was located on LG1, and probably corresponds to the S locus. We show that the T locus, a relic of a multilocus system, functions through interaction with the S locus: F2 segregation of incompatibility phenotypes and linked markers demonstrated that the S/t pollen genotype combination, expected to be compatible on selfing, was sometimes incompatible. Further evidence is presented to show that this interaction must be dependent on yet another locus located on LG2. A prime candidate would be the Z incompatibility locus.