The pannexin (Px) and connexin (Cx) families form multimeric hemichannels that mediate cellular transport of a wide variety of signaling and other molecules and exhibit pathophysiological and pharmacological functions. Twenty-four Px and Cx subtypes have been identified in humans and 23 in mice. The purpose of this study is to establish a quantitative protein atlas of Px and Cx subtypes in mouse and human tissues and cancer cell lines by means of quantitative targeted absolute proteomics, using an internal standard protein in which stable-isotope-labeled target peptides selected according to in silico criteria are concatenated together with internal reference peptides for the determination of the protein amount. This quantification system enabled us to cover 20 of 24 subtypes (83%) in humans, and 21 of 23 subtypes (91%) in mice. In mice, Px1, Cx32, and Cx43 were most abundantly expressed in the small intestine, liver and pancreas, and brain capillary, brain, and heart, respectively. Human blood-brain barrier endothelial cells (human cerebral microvessel endothelial cells) highly expressed Px1 and Cx43. Among human cancer cells, Panc-1 selectively expressed Px1, and Caco-2 cells abundantly expressed Cx32, while MCF-7 and AsPC-1 did not express any subtypes of hemichannels tested. These results suggest that Px1, Cx32, and Cx43 appear to play predominant roles in normal tissues and some cancer cells.,Copyright ? 2020 American Pharmacists Association?. Published by Elsevier Inc. All rights reserved.