1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline. Inhibitors of the type III phosphodiesterase isoenzyme (SK&F 94120 and SK&F 94836) were without effect upon basal or isoprenaline-stimulated cyclic AMP responses in these cells.5. Carbachol (1 nM-I 00 microM) produced concentration-dependent inhibition of the [3H]-cyclic AMP response to 1 microM isoprenaline in human cultured tracheal smooth muscle cells (IC50 0.24 JM). Carbachol(1 JM) inhibited the [3H]-cyclic AMP response to 1 JM isoprenaline by 60%. This effect of carbachol was itself inhibited by atropine (50 nM) (KA 2.3 x 109 M-') indicating the involvement of a muscarinic receptor.6. These results show that primary cultures of human tracheal smooth muscle cells demonstrate cyclic AMP responses to direct receptor stimulation, adenylyl cyclase activation and inhibition with nonselective and type IV-selective cyclic AMP phosphodiesterase isoenzyme inhibitors, and that the cyclic AMP response to isoprenaline can be inhibited by muscarinic receptor stimulation.