Gap junction-mediated intercellular communication in untreated and phenobarbital-treated C57BL/6 X C3H F1 mouse hepatocytes was evaluated by microinjection of fluorescent Lucifer Yellow CH dye. Intercellular communication (dye coupling) was detected in untreated hepatocytes after 0.5 h in culture, reached a maximum level in 24- and 48-h-old cultures (85.2%), and then decreased over the next 72 h. Phenobarbital (20-500 micrograms/ml) decreased dye coupling in a dose-related manner when added to freshly plated cultures. This inhibitory effect was evident during 0.5-12 h of treatment but was not seen in cultures treated for 24 h. Phenobarbital also decreased dye coupling within 30 min when added to established (24-h-old) hepatocyte cultures. This effect was maximal after 2 h treatment. In these cultures, dye coupling recovered within 15 min after removal of the promoter. Hepatocytes, pretreated with phenobarbital for 24 h, did not show inhibition of dye coupling after reapplication of phenobarbital. Thus, phenobarbital inhibited mouse hepatocyte dye coupling rapidly and reversibly, and the cells became refractory to the inhibitory effect after prolonged treatment.