In order to investigate the generation of conotoxin diversity, delta-conotoxin sequences from nine Conus species were analyzed in the context of their phylogeny. Using a standard molecular marker, mitochondrial 16S RNA, we determined that the delta-conotoxins were derived from three distinct species clades based on the phylogenetic reconstruction of a large set (>80) of Conus species and other toxoglossate molluscs. Four different mechanisms appear to have contributed to the diversity of the delta-conotoxins analyzed: (1) Speciation: Delta-conotoxins in different species diverge from each other (the prepro regions of orthologous genes somewhat more slowly than the reference rRNA rate, the mature toxin regions significantly faster). (2) Duplication: Intraspecific delta-conotoxin divergence is initiated by gene duplication events, some of which may have predated the species itself. (3) Recombination: A novel delta-conotoxin may arise through recombination of two parental delta-contoxin genes. (4) 'Focal hypermutation': This sudden, almost saltatory change in sequence is always restricted to the mature toxin region. The first three have been recognized previously as mechanisms important for the evolution of gene families in other phylogenetic systems; the last is a remarkable, mechanistically unexplained and specialized feature of Conus peptide diversification.