1. Nitric oxide (NO) is known from previous studies to be the principle transmitter in NANC inhibitory nerves supplying the hamster urethra. However, the identity of the cotransmitter(s) responsible for the responses remaining following block with L-NG-nitroarginine methyl ester (L-NAME) is not known. 2. Electrical field stimulation (EFS) of circular strips of hamster proximal urethra precontracted with arginine vasopressin (AVP 10(-8) M), and in the presence of phentolamine (10(-6) M), propranolol (10(-6) M) and atropine (10(-6) M), caused frequency-dependent relaxation, which was attenuated by suramin (10(-4) M) and reactive blue 2 (RB2; 2 x 10(-4) M), but not by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10(-4) M), alpha-chymotrypsin (10-50 u ml(-1)) or by the vasoactive intestinal polypeptide (VIP) antagonist, [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP, (5 x 10(-7)-10(-6) M). In the presence of indomethacin (10(-6) M) frequency-dependent relaxations to EFS were enhanced, particularly at the lower frequencies of stimulation. EFS-induced relaxation was blocked by tetrodotoxin (10(-6) M), indicating its neurogenic origin. 3. Exogenous ATP (10(-7)-10(-3) M) produced concentration-related relaxations which were attenuated by the P2-purinoceptor antagonists suramin (10(-4) M) and RB2 (2 x 10(-4) M) but not by PPADS (10(-4) M). ATP-induced relaxations were also reduced significantly by indomethacin (10(-6) M). The inhibitory responses to ATP were urothelium- and NO-independent, since they were not affected by either removal of urothelium or by L-NAME (10(-4) M). 4. Exogenous VIP (10(-9)-10(-7) M) induced concentration-related relaxations which were not affected by urothelium removal, L-NAME (10(-4) M), alpha-chymotrypsin (10-50 u ml(-1)) or by [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (3 x 10(-7)-10(-6) M). Nevertheless, suramin (10(-4) M) and RB2 (2 x 10(-4) M) but not PPADS (10(-4) M) antagonized the VIP-induced relaxant responses. Calcitonin gene-related peptide (CGRP: 10(-9)-10(-7) M) was devoid of any effect or only elicited a small relaxant response in AVP-precontracted strips. 5. Exogenous prostaglandin E2 (PGE2; 10(-9)-3 x 10(-6) M) and the NO donor, sodium nitroprusside (SNP; 10(-8)-3 x 10(-5) M) elicited concentration-related relaxations on the hamster proximal urethra which were not attenuated by suramin (10(-4) M), RB2 (2 x 10(-4) M), or by PPADS (10(-4) M), indicating a specific inhibitory effect of the antagonists used. 6. In summary, these results are consistent with the view that ATP is an inhibitory transmitter released from inhibitory nerves supplying the NANC relaxation of hamster proximal urethra. The relaxant effect of ATP is NO- and urothelium-independent. The present study did not demonstrate whether VIP is released from parasympathetic nerves during EFS, since both alpha-chymotrypsin and [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP were ineffective on neurogenic responses.