Self-incompatibility in the Solanaceae is mediated by S-RNase alleles expressed in the style, which confer specificity for pollen recognition. Nicotiana alata has been successfully used as an experimental model to elucidate cellular and molecular aspects of S-RNase-based self-incompatibility in Solanaceae. However, S-RNase alleles of this species have not been surveyed from natural populations and consequently the S-haplotype diversity is poorly known. Here the molecular and functional characterization of seven S-RNase candidate sequences, identified from a natural population of N. alata, are reported. Six of these candidates, S ( 5 ), S ( 27 ), S ( 70 ), S ( 75 ), S ( 107 ), and S ( 210 ), showed plant-specific amplification in the natural population and style-specific expression, which increased gradually during bud maturation, consistent with the reported S-RNase expression. In contrast, the S ( 63 ) ribonuclease was present in all plants examined and was ubiquitously expressed in different organs and bud developmental stages. Genetic segregation analysis demonstrated that S ( 27 ), S ( 70 ), S ( 75 ), S ( 107 ), and S ( 210 ) alleles were fully functional novel S-RNases, while S ( 5 ) and S ( 63 ) resulted to be non-S-RNases, although with a clearly distinct pattern of expression. These results reveal the importance of performing functional analysis in studies of S-RNase allelic diversity. Comparative phylogenetic analysis of six species of Solanaceae showed that N. alata S-RNases were included in eight transgeneric S-lineages. Phylogenetic pattern obtained from the inclusion of the novel S-RNase alleles confirms that N. alata represents a broad sample of the allelic variation at the S-locus of the Solanaceae.