In virtually all species examined to date, the functionally active third component of complement (C3) is encoded by a single gene. We have recently demonstrated, however, that trout possess three structurally and functionally distinct active C3 that represent the products of at least two different C3 genes. In the present study, we provide evidence that multiple forms of functional C3 occur not only in the rainbow trout (Salmo gairdneri), a quasi-tetraploid old teleost fish, but also in the diploid gilthead sea bream (Sparus aurata), a modern teleost fish. In the gilthead sea bream, we have characterized five different forms of C3 (C3-1, C3-2, C3-3, C3-4, and C3-5); in addition, we have identified and isolated a C5-like molecule. Each of the six proteins was composed of an alpha-chain and a beta-chain; however, only the C3 isoforms contained a thioester bond in their alpha-chains. These proteins all differed in the molecular masses of their alpha- and beta-chains and in their glycosylation patterns, reactivity with various Abs, tryptic peptide maps, and NH2-terminal sequences of their chains. These observations together with the fact that each of the six proteins were also purified from a single fish suggest that the C3 isoforms represent the products of several genes. The presence of multiple forms of C3 in a modern diploid fish, very distant in evolutionary time from the trout, strongly suggests that the C3 isoforms generated once from a single C3 gene have remained functional in the genomes of these animals. These findings not only have important consequences for our understanding of the evolution of the C3 protein, but also provide evidence for the formation and generation of a new C3-related gene family.