Ral GDP dissociation stimulator (RalGDS) and its family members RGL, RLF and RGL2 are involved in Ras and Ral signaling pathways as downstream effector proteins. Here we report the precise localization and cloning of two forms of human RGL gene differing at the amino terminus. Transcript A, cloned from liver cDNA libraries has the same amino terminus as the mouse RGL, whereas transcript B cloned from brain has a substitution of 45 amino acids for the first nine amino acids. At the genomic level, exon 1 of transcript A is replaced by two alternative exons (1B1 and 1B2) in transcript B. Both forms share exons 2 through 18. The human RGL protein shares 94% amino acid identity with the mouse protein. Northern blot analysis shows that human RGL is expressed in a wide variety of tissues with strong expression being seen in the heart, brain, kidney, spleen and testis.