The scrape loading/dye transfer technique was used to analyse whether gap junction coupling of endothelial cells (EC) and vascular smooth muscle cells (SMC) is affected by dipyridamole. We found that presence of dipyridamole (1–50 mM) for 24 h increased the gap junction coupling in both cell types. In EC, the gap junction coupling was already affected at 1 mM dipyridamole while the SMC needed a concentration of 5 mM for a significant increase of gap junction coupling. The analysis of the time course at which dipyridamole affected the gap junction coupling showed that a minimal incubation time of 3 and 6 h was necessary for SMC and EC, respectively. With respect to the mechanisms behind the effect of dipyridamole on the gap junction coupling, it was found that protein kinase A (PKA) inhibitors (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) or Rp-cAMPS) antagonised the dipyridamole induced potentiation of gap junction coupling whereas forskolin or membrane permeable cAMP analogues (dibutyryl-cAMP or 8-bromo-cAMP) increased the gap junction coupling similar to dipyridamole. The results indicate that dipyridamole probably increases the gap junction coupling of the vascular cells by a cAMP/PKA dependent pathway. Moreover, the results identify the gap junction coupling of the vascular system as a therapeutic target in treatment of vascular impairment.