Connexins make up a gene family encoding proteins that form intercellular channels known as gap junctions. Decreases in connexin expression and loss of intercellular communication have been associated with the malignant phenotype in some animal and human cells. The expression of connexin 26 and 43 mRNA was evaluated in cultured normal and malignant human urothelial cells. The normal urothelial cells were shown by Northern analysis to express both connexins. Increased confluence of the cultured normal human urothelial cells was associated with upregulation of connexin 26 mRNA. Connexin 26 mRNA expression was decreased in the bladder cancer cells. Using a human connexin 26 complementary DNA probe, nuclear run-on assays demonstrated that the decreased expression in the cancer cells was due to a failure of transcription. Southern blot analysis did not reveal any alterations in the genomic DNA. Assessment of gap junction function by scrape loading of lucifer yellow demonstrated dye transfer in normal urothelial cells but not in bladder cancer cells. Downregulation of connexin 26 mRNA was associated with functional loss of intercellular communication in the human bladder cancer cells. Connexin 43 expression varied considerably in the bladder cancer cell lines and did not correlate with dye transfer of lucifer yellow. These data suggest that alterations in the regulation of connexin 26 expression are associated with and may contribute to the malignant phenotype in bladder cancer.