Parasympathetic nerves provide the major excitatory innervation to the detrusor muscle of the urinary bladder, but it was shown as long ago as the 19th century that a substantial component of the neurotransmission is atropine-resistant (Langley & Anderson, 1895). Subsequently, desensitisation of P2X₁ receptors by α,β-meATP was found to abolish the non-cholinergic component, identifying ATP as a cotransmitter with acetylcholine (Kasakov & Burnstock, 1983). The aim of this study was to characterise the effects of P2X receptor antagonists on the non-cholinergic component of neurotransmission. Adult, male Dunkin Hartley guinea-pigs were humanely killed and longitudinal strips (12 mm x 3 mm) of urinary bladder mounted under isometric conditions in 2 ml baths at 35°C. Intramural nerves were stimulated by electrical field stimulation (EFS) at 4 Hz, 0.15 ms pulse width, for 20 s at 10 min intervals, via platinum wire electrodes. Atropine (1 μM) and prazosin (100 nM) were present throughout. Data are expressed as mean ± s.e.m. or geometric mean with 95% confidence limits (95% cl), and were compared by Student’s t test, or one-way ANOVA and Tukey’s comparison as appropriate. Concentration-inhibition response curves were fitted to the data by logistic (Hill equation), non-linear regression analysis. Atropine (1 μM) abolished contractions evoked by exogenous acetylcholine (10 μM) (n=4), but reduced the peak amplitude of responses to 4 Hz EFS by only 27.7 ± 7.9% (n=6). PPADS (0.1-100 μM) and suramin (1-300 μM) inhibited the atropine-resistant contractions with IC₅₀ (95% cl) values of 6.9 μM (0.7-78.9 μM, n=5) and 13.8 μM (7.9-24.1 μM, n=6) respectively, but 30-40% of the response remained at the highest concentrations used. Tetrodotoxin (1 μM) abolished the remaining responses. PPADS and suramin also inhibited contractions elicited by exogenous ATP (300 μM) and α,β-meATP (1 μM), with potencies similar to those against 4Hz EFS (n=4-8). Whilst the responses to α,β-meATP were abolished, 30-40% of the peak response to ATP was not. The atropine-resistant neurogenic contractions were also only partially inhibited by 100 μM of the P2X antagonists NF279 (31.9 ± 3.7% decrease, n=7), MRS2159 (57.0 ± 4.4% decrease, n=5) and reactive blue 2 (32.8 ± 5.2% decrease, n=6), all of which abolished contractions evoked by α,β-meATP (1 μM). In contrast, desensitisation of P2X₁ receptors by administration of α,β-meATP (50 μM) abolished responses to 4 Hz EFS (n=12), α,β-meATP (1 μM, n=4) and ATP (300 μM, n=5). Thus, whilst the non-cholinergic component of neurotransmission is abolished by desensitisation of the P2X₁ receptor by α,β-meATP, it is only partially inhibited by P2X₁ antagonists. At present the mechanism underlying the atropine- and P2X₁ receptor antagonist-resistant component of parasympathetic neurotransmission is unclear.