Two isozymes of cGMP-dependent protein kinase (cGMP kinase) have been identified. Polyclonal antibodies were developed which recognize both isozymes or specifically the I alpha and I beta isoform. The specificity of these antibodies was verified by using the recombinant or purified I alpha and I beta isozymes. The antibodies cross-reacted with the purified isozymes of cGMP kinase from bovine tracheal smooth muscle. The tissue concentration of cGMP kinase was determined by ELISA. High concentrations (greater than 10 pmol/g wet tissue) were present in bovine lung, rumen, trachea, aorta, uterus and stomach. The tissue distribution of the isozymes I alpha and I beta was investigated by immunoblots using crude extracts of the different tissues. The I beta-specific antibody yielded strong signals with extracts of trachea, aorta, stomach and uterus, whereas heart, cerebellum and lung apparently contain mainly the I alpha isozyme.