Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic [c]GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cGMP levels were detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cGMP levels were detected during carbachol-induced contractions, i.e., guinea pig myometrium and taenia coli, the contractions appeared to precede the cGMP increases by several seconds. No significant changes in cAMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.