Hepatocytes prepared from 18-day-old mouse embryos were grown in serum-free medium and reached confluence after two days in culture. The total amount of the 26 kDa gap junction protein decreased in these cells during the first 24 h in culture and increased again between day 1 and day 3 more than 10-fold. At day 3 a half-life time of 2.5 to 3 h was determined for the 26 kDa protein by [35S]methionine incorporation and immunoprecipitation using affinity-purified anti-26 kDa. Incorporation of [32P]orthophosphate into the 26 kDa protein of cultured hepatocytes was found at serine residues (98%) and tyrosine residues (about 2%). The addition of dibutyryl cyclic adenosine monophosphate (db cAMP) to the culture medium at day 2 had two effects: After 15 min the extent of phosphorylation of the 26 kDa protein increased 2.7-fold whereas the total amount of the 26 kDa protein increased only 1.2-fold. After 3 h of incubation with db cAMP, a 2.5-fold increase of the 26 kDa protein was noticed which was accompanied by a 3.2-fold increase in phosphorylation of serine residues. The effects of db cAMP on phosphorylation of the 26 kDa protein could be augmented or mimicked by the addition of isoproterenol, theophylline or forskolin to the culture medium of hepatocytes. In extracts of rat hepatocarcinoma MH1C1 cells and dog kidney MDCK cells, a phosphorylated 26 kDa protein can be immunoprecipitated using anti-liver 26 kDa. These results demonstrate that the gap junction 26 kDa protein can be posttranslationally modified by cAMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)