Although Petunia axillaris subsp. axillaris is described as a self-incompatible taxon, some of the natural populations we have identified in Uruguay are composed of both self-incompatible and self-compatible plants. Here, we studied the self-incompatibility (SI) behavior of 50 plants derived from such a mixed population, designated U83, and examined the cause of the breakdown of SI. Thirteen plants were found to be self-incompatible, and the other 37 were found to be self-compatible. A total of 14 S-haplotypes were represented in these 50 plants, including two that we had previously identified from another mixed population, designated U1. All the 37 self-compatible plants carried either an SC1- or an SC2-haplotype. SC1SC1 and SC2SC2 homozygotes were generated by self-pollination of two of the self-compatible plants, and they were reciprocally crossed with 40 self-incompatible S-homozygotes (S1S1 through S40S40) generated from plants identified from three mixed populations, including U83. The SC1SC1 homozygote was reciprocally compatible with all the genotypes examined. The SC2SC2 homozygote accepted pollen from all but the S17S17 homozygote (identified from the U1 population), but the S17S17 homozygote accepted pollen from the SC2SC2 homozygote. cDNAs encoding SC2- and S17-RNases were cloned and sequenced, and their nucleotide sequences were completely identical. Analysis of bud-selfed progeny of heterozygotes carrying SC1 or SC2 showed that the SI behavior of SC1 and SC2 was identical to that of SC2 and SC2 homozygotes, respectively. All these results taken together suggested that the SC2-haplotype was a mutant form of the S17-haplotype, with the defect lying in the pollen function. The possible nature of the mutation is discussed.