Mardin Darby canine kidney (MDCK) cells are widely used as a model of renal epithelial ion transport. In previous work, we found that iodide efflux by type I MDCK cells was activated by cAMP and Ca²⁺ agonists and cell swelling, whereas that of type II MDCK cells was activated by cell swelling only (Li et al 2003). These data suggest that MDCK cells possess multiple different types of Cl^– channels. To test this hypothesis, in the present study, we used RT-PCR to explore the expression of Cl^– channels in MDCK cells. We investigated the expression of (i) CLCN2 and CLCN3, two members of the CLC family of Cl^– transporters, (ii) CFTR, the cAMP-stimulated Cl^– channel, (iii) bestrophin, the Ca²⁺-activated Cl^– channel and (iv) TTYH2 and TTYH3, putative Ca²⁺– and cell swelling regulated Cl^– channels. We extracted total RNA from MDCK cells using RNAqueous isolation kit (Ambion). With DNAse treated RNAs, we synthesized and amplified cDNAs using the One Step RT-PCR kit (Qiagen). We designed primers for RT-PCR using Primer 3 software based on consensus sequences across species. We visualised DNA fragments after agarose gel electrophoresis by staining with ethidium bromide. Then, we purified RT-PCR products with the QIAquick PCR purification kit before sequencing the DNA fragments using the MWG nucleotide sequencing service. As controls, we examined the expression of β actin and performed reactions without a reverse transcription step to monitor contamination of samples by genomic DNA. Expression of each gene was examined in multiple samples from both cell types. Both type I and type II MDCK cells expressed each of the different genes investigated. However, in the case of bestrophin, although DNA fragments of the expected size were obtained, the sequence of these fragments was not verified by nucleotide sequencing. With the exception of TTYH2, which was more abundant in type II MDCK cells, visual inspection of agarose gels suggested that each of the different genes examined were equally expressed in type I and type II MDCK cells. These data contrast strikingly with our functional data, which suggest that type I, but not type II, MDCK cells express Cl^– channels regulated by cAMP-dependent phosphorylation and the intracellular Ca²⁺ concentration. Future studies should explore the reason for the dichotomy between the molecular and functional expression of Cl^– channels in MDCK cells.