ADP-ribosylation factor 4 (ARF4) is a member of a family of approximately 20 kDa guanine nucleotide-binding proteins that were initially identified by their ability to stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. They have recently been shown to play a role in vesicular trafficking and as activators of phospholipase D. The organization of the human ARF4 gene was determined from a genomic clone isolated from an arrayed PAC genomic library. The gene spans approximately 12 kb and contains six exons and five introns. Translation initiates in exon 1 and terminates in exon 6. Nuclease protection experiments indicated that the major transcription initiation site is located 211 bp 5' to the start of translation. In some cell lines derived from human tissues, however, multiple initiation sites were observed. The proximal 5'-flanking region of the human ARF4 gene lacks a TATA box, is highly GC rich, and contains multiple potential Spl-binding sites. An alignment of the exons for the class I ARF genes (ARF1, ARF2, and ARF3) and class II ARF genes (ARF4 and ARF5) reveals that the members of each class share a common gene organization. The structures of the class I and II ARF genes, however, are quite distinct and support the division of the ARFs into these groups based on deduced amino acid sequence, protein size, phylogenetic analysis, and gene structure.