The effects of an increase in intracellular cyclic GMP (cGMP)-concentration on gap junctional current (Ij) were studied in cultured neonatal rat heart cells using both the whole-cell and perforated patch voltage-clamp method. In whole-cell measurements, exposure to 8-bromo-cGMP or carbachol reduced Ij. With the perforated patch technique, on the other hand, Ij was not affected by either 8-bromo-cGMP or carbachol. Addition of alkaline phosphatase prevented the carbachol-induced decrease in Ij in whole-cell measurements. Reduction of Ij in well-coupled cell pairs by application of heptanol allowed us to study the effects of these substances on the single gap junction channel level. We found that cGMP-treatment shifts the single channel conductance (gamma j) from 43 to 21 pS in whole-cell measurements and that intracellular addition of phosphatase prevents this shift. In contrast, intracellular phosphatase-treatment itself shifts gamma j to 70 pS. Our results indicate that Cx43-gap junction channels may exhibit three conductance levels (21 pS, 40-45 pS and 70 pS), depending on the phosphorylation state of the protein.