Pannexins are mammalian orthologs of the invertebrate gap junction proteins innexins and thus have been proposed to play a role in gap junctional intercellular communication. Localization of exogenously expressed pannexin 1 (Panx1) and pannexin 3 (Panx3), together with pharmacological studies, revealed a cell surface distribution profile and life cycle dynamics that were distinct from connexin 43 (Cx43, encoded by Gja1). Furthermore, N-glycosidase treatment showed that both Panx1 (approximately 41-48 kD species) and Panx3 (approximately 43 kD) were glycosylated, whereas N-linked glycosylation-defective mutants exhibited a decreased ability to be transported to the cell surface. Tissue surveys revealed the expression of Panx1 in several murine tissues--including in cartilage, skin, spleen and brain--whereas Panx3 expression was prevalent in skin and cartilage with a second higher-molecular-weight species present in a broad range of tissues. Tissue-specific localization patterns of Panx1 and Panx3 ranging from distinct cell surface clusters to intracellular profiles were revealed by immunostaining of skin and spleen sections. Finally, functional assays in cultured cells transiently expressing Panx1 and Panx3 were incapable of forming intercellular channels, but assembled into functional cell surface channels. Collectively, these studies show that Panx1 and Panx3 have many characteristics that are distinct from Cx43 and that these proteins probably play an important biological role as single membrane channels.