ABSTRACT: Human RCAN3 (regulator of calcineurin 3) belongs to the human RCAN gene family.In this study we provide, with in silico and in vitro analyses, the first detailed description of the human multi-transcript RCAN3 locus. Its analysis revealed that it is composed of a multigene system that includes at least 21 RCAN3 alternative spliced isoforms (16 of them identified here for the first time) and a new RCAN3 antisense gene (RCAN3AS). In particular, we cloned RCAN3-1,3,4,5 (lacking exon 2), RCAN3-1a,2,3,4,5, RCAN3-1a,3,4,5, RCAN3-1b,2,3,4,5, RCAN3-1c,2,3,4,5, RCAN3-1c,2,4,5 and RCAN3-1c,3,4,5, isoforms that present a different 5' untranslated region when compared to RCAN3. Moreover, in order to verify the possible 5' incompleteness of previously identified cDNA isoforms with the reference exon 1,