1. The activity of rat alpha and beta calcitonin gene-related peptide (CGRP) as compared to the structurally related peptide, rat amylin, has been investigated in the guinea-pig isolated left atrium (electrically driven), in mucosa-free strips from the base of the guinea-pig urinary bladder and in the rat isolated vas deferens (pars prostatica). The antagonist activity of the C-terminal fragment of human alpha CGRP, alpha CGRP(8-37), was also investigated. 2. In the guinea-pig isolated left atrium the three peptides produced a concentration-related positive inotropic effect, amylin being about 16 and 31 times less potent than alpha or beta CGRP, respectively. Human alpha CGRP(8-37) produced a rightward displacement of the log concentration-response curve to the three agonists tested, without depression of maximal response attainable. Apparent pKB values calculated on the basis of the displacement produced by 1 microM human alpha CGRP(8-37) indicated an agonist-independent affinity of the antagonist (6.66 +/- 0.11 for alpha CGRP, 6.42 +/- 0.17 for beta CGRP and 6.95 +/- 0.11 for amylin). 3. In the guinea-pig isolated urinary bladder, alpha or beta CGRP or amylin produce a concentration-related inhibition of twitch contractions evoked by train electrical field stimulation (10 Hz frequency, 0.25 ms duration at 100 V for 0.5 s every 60 s). Amylin was about 100 times less potent than alpha or beta CGRP. Human alpha CGRP(8-37) (3 microM) did not significantly affect the inhibitory action of the three agonists tested.4. In the rat isolated vas deferens, a or PCGRP or amylin produced a concentration-related inhibition of twitch contractions evoked by electrical field stimulation (0.2 Hz frequency, 0.5 ms duration at 60 volts). Amylin was about 100 times less potent than a or PCGRP. Human aCGRP(8-37) at 3 microM did not significantly affect the inhibitory action of amylin and at 3 microM antagonized the responses to rat a and PCGRP with apparent pKB values of 5.86 +/- 0.15 and 6.11 +/- 0.13, respectively.5. These findings indicate that multiple receptors mediate the actions of peptides of the CGRP/amylin family in the preparations investigated. In the guinea-pig atrium both a and P forms of rat CGRP as well as amylin act by stimulating a single class of receptors which are sensitive to the inhibitory action of human aCGRP(8-37). In rat isolated vas deferens, at least two receptors could be present, one activated by a and PCGRP and partially sensitive to human aCGRP(8-37) and another which is sensitive to amylin but not recognised by human aCGRP(8-37). This latter type of receptor could be entirely responsible for the action of the agonists in the guinea-pig urinary bladder.