(1) This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). (2) Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. (3) Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. (4) Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. (5) The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. (6) This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). (7) Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. (8) Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid.