T1 a is a protein of unknown function that is expressed at the plasma membrane in epithelia involved in fluid transport, including type I alveolar epithelial cells, choroid plexus, and ciliary epithelium. The purpose of this study was to test the hypothesis that T1 a functions as a water channel or a regulator of aquaporin-type water channels that are coexpressed with T1 a. Two complementary DNAs (cDNAs)(hT1a-1 and hT1 a-2) encoding human isoforms of T1 a were cloned by homology to the rat T1 a coding sequence. The cDNAs encoded 164 (hT1 a-1) and 162 (hT1 a -2) amino acid proteins with high homology to rat T1 a in a putative membrane-spanning domain. hT1 a-1 transcripts of 2.6 and 1.4 kb were detected in human lung, heart, and skeletal muscle, and a single hT1 a-2 transcript of 1.2 kb was detected in human lung. Rat and mouse T1 a were isolated by reverse transcription-polymerase chain reaction and confirmed by DNA sequence analysis. Expression of mouse, rat, and human T1 a isoforms in Xenopus oocytes did not increase osmotic water permeability (Pf) above that in water-injected oocytes, nor was there an effect of protein kinase A or C activation; Pf was increased. 10-fold in positive control oocytes expressing aquaporin (AQP)1 or AQP5. Coexpression of AQP1 or AQP5 with excess T1a gave P f not different from that in oocytes expressing AQP1 or AQP5 alone. Oocyte plasma membrane localization of epitope-tagged T1 a protein was confirmed and quantified by immunoprecipitation of microdissected plasma membranes. Quantitative densitometry indicated that the single-channel water permeability of T1 a is under 23102 16cm 3/s, suggesting that T1 a is not involved in the high transalveolar water permeability in intact lung. The cloning of hT1 a isoforms may permit the development of an assay of type I cell antigen in airspace fluid as a marker of human lung injury. Ma, T., B. Yang, M. A. Matthay, and A. S. Verkman. 1998. Evidence gainst a role of mouse, rat, and two cloned human T1 a isoforms as a water channel or a regulator of aquaporin-type water channels. Am. J. Respir. Cell Mol. Biol. 19: 143-149.