Chimpanzee, tamarin, and marmoset interleukin-3 (IL-3) genes were cloned, sequenced, and expressed. Western blot analysis demonstrated that functional genes were isolated. IL-3 sequences were compared with those of mouse, rat, rhesus monkey, gibbon, and man. Multiple alignment of the IL-3 coding regions showed that only a few regions had been conserved during mammalian evolution, which are likely associated with functional domains of the IL-3 protein. Substitution rates for the various lineages were calculated and the numbers of synonymous and nonsynonymous substitutions were estimated separately. Distance matrices of the IL-3 coding regions were used to construct phylogenetic trees which revealed large differences in IL-3 evolution rate as well as a more rapid substitution rate for rodents and a rate slowdown during hominoid evolution. Extremes were rhesus monkey IL-3, which accumulated few synonymous substitutions, and gibbon IL-3, which had almost exclusively synonymous substitutions. In rhesus monkey IL-3, nonsynonymous substitutions outnumbered synonymous substitutions, which could not be readily explained by a random process of substitutions. We assume that during evolution of IL-3, the majority of the amino acid replacements and the impaired interspecies functional cross-reactivity originate from selection mechanisms with the most likely selective force being the structure of the heterodimeric IL.3 cell-surface receptor. Insight into IL-3 architecture and structural analysis of the IL-3 receptor are needed to analyze the unusually fast evolution of IL-3 in more detail.