The organization of the insulin gene of the owl or night monkey (Aotus trivirgatus), a New World primate, is similar to that of the human gene. The sequences of these two genes and flanking regions possess 84.3% homology. An unusual feature of the owl monkey gene is the partial duplication and insertion of a portion of the A-chain coding sequence into the 3' untranslated region. The insulin gene of this primate also lacks a region of tandem repeats that is present in the 5' flanking region of the human and chimpanzee genes. Owl monkey preproinsulin has 85.5% identity with the human insulin precursor and is the most divergent of the primate insulins/preproinsulins yet described. The differences between owl monkey and human preproinsulin include three substitutions in the signal peptide, two in the B chain, seven in the C peptide, and three in the A chain. One of these replacements is the conservative substitution of valine for isoleucine at position A2, an invariant site in all other vertebrate insulins and insulin-like growth factors. The substitutions in owl monkey insulin at B9, B27, A2, A4, and A17 alter its structure so that it has only 20% of the receptor-binding activity and 1% of the affinity with guinea pig anti-porcine insulin antibodies as compared to human insulin.