Little is known about whether culturing corneal limiting layers causes changes in the expression of their membrane transporter proteins from those present in fresh tissues. Accordingly, we compared mRNA abundance of three well-described types of transporters: water channel aquaporin 1 (AQP1), glucose transporter (GLUT1), and Na:K:2Cl cotransporter (NKCC), as well as NKCC protein levels in fresh bovine corneal epithelium and endothelium with those in their cultured counterparts. Abundance of mRNA encoding AQP1, GLUT1, and NKCC was quantified by a lysate nuclease protection assay. NKCC transcription was further characterized by Northern blotting. All data were normalized to cell DNA and protein contents. In the fresh epithelium, in all three cases mRNA levels were two to four times higher than in the endothelium. Expression of AQP1 and GLUT1 was 10 to 12 times higher than that of NKCC. After the third passage, the endothelial cell mRNA abundance in each case decreased 2- to 3-fold. Passage-dependent decreases were also observed in NKCC protein expression in the epithelial cells. In both corneal layers, there was a qualitative correlation between NKCC mRNA and protein levels. Both in fresh and cultured epithelial and endothelial cells, a shark NKCC1 DNA probe hybridized with mRNAs of two different lengths (about 5.0-5.5 and 7.0-7.5 kb). An anti-NKCC T4 monoclonal antibody recognized two major proteins with apparent molecular masses of 190 to 200 and 150 to 160 kDa. In summary, membrane transporter function in culture may not be always indicative of their role in fresh tissue since in cultured cells AQP1, GLUT1, and NKCC mRNA levels declined. Furthermore, in both epithelial and endothelial cells, there is expression of two different proteins and mRNAs that possibly encode for secretory (NKCC1) and absorptive (NKCC2) isoforms.