Two neurotransmitters, ACh and ATP, initiate nerve-mediated contractions in detrusor smooth muscle from guinea-pigs, and humans with bladder over-activity; whereas only ACh has a role in muscle from stable human bladders (Bayliss et al., 1999). One hypothesis for this difference is that in the latter group ATP is released but completely broken down by ectonucleotidases whereas in the former tissues hydrolysis is only partial (Harvey et al., 2002). We have further tested this hypothesis by examining the effects of apyrase (a non-specific nucleotidase) and ARL 67156 (an ectonucleotidase inhibitor) on contractile performance in guinea-pig and human detrusor.Detrusor strips were obtained from guinea-pigs, killed by Schedule I methods, and humans, with ethical committee approval, with urodynamically over-active or stable bladders. Preparations were superfused with a HEPES-buffered solution (pH7.3 at 37°C). Tetrodotoxin-sensitive (1µM) nerve-mediated contractions were elicited by tetanic electrical field stimulation (0.1 ms pulses, 1-40Hz). Data are shown as mean ± s.d., (n) is number of experiments. Differences between data sets were tested for significance (p<0.05) by Student’s t-test.Apyrase (10U.ml^-1) attenuated significantly nerve-mediated contractions in strips from guinea-pigs (to 82.4±15.4% control, n=16) and overactive human bladders (86.7±7.2% control, n=4). Apyrase had no significant effect on contractions in strips from stable bladders (94.6±15.0% control, n=3). The frequency-dependence of contraction strength was unaffected by apyrase in the former groups (half-maximal frequency: guinea-pig 5.6±2.0 vs 7.0±3.7 Hz; human over-active 19.8±10.2 vs 15.4±9.4 Hz). The purinergic component of contraction was measured as the residual contraction in 1µM atropine and was; guinea-pig 47.3±13.7% of control; human over-active 83.6±10% and; human stable 100±0.0%. ARL 67156 (100µM) enhanced guinea-pig contractions at 16Hz stimulation by 52.9±12.7%.The data show a correlation between the effect of apyrase on nerve-mediated contractions in human and guinea-pig detrusor and the proportion of atropine resistance. This is consistent with the hypothesis that ATP contributes to nerve-mediated contractions in guinea-pig and over-active human bladders by incomplete breakdown prior to its activation of detrusor smooth muscle. This was corroborated by an enhancement of contraction strength in guinea-pig tissue by ARL 67156, which would have reduced further ATP breakdown. The lack of effect on the shape of the force-frequency response implies apyrase has no significant effect on transmitter release per se.