Abstract Background Hypertonicity of the internal anal sphincter (IAS) appears to be involved in the pathogenesis of anal fissure. The relaxant effects of sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, on isolated human IAS were investigated. Methods The efficacy (maximal effect, Emax) and potency (−log IC50, where IC50 is half-maximal inhibitory concentration) of the PDE5 inhibitors, sildenafil and zaprinast, and of nitric oxide donors, sodium nitroprusside and glyceryl trinitrate, as relaxants of histamine (0·1 mmol/l)-induced tone were examined in IAS strips under isometric contraction. The presence of PDE5 isoenzymes and changes in intracellular calcium and cyclic nucleotide levels in IAS muscle were tested by real-time reverse transcriptase–polymerase chain reaction, epifluorescence microscopy and enzyme immunoassay respectively. Results Sildenafil produced a concentration-related inhibition of the mean(s.e.m.) histamine-induced tone (Emax 83(2) per cent, − log IC50 7·04(0·05); n = 12). Zaprinast produced relaxation to similar degree, but with lower potency. Nitric oxide donors also relaxed IAS. Sildenafil (1 µmol/l) produced a 1·8-fold increase in guanosine 3′,5′-cyclic monophosphate content, with no change in adenosine 3′,5′-cyclic monophosphate levels. Sildenafil markedly depressed the peak intracellular calcium increase evoked by histamine. PDE5A1, PDE5A2 and PDE5A3 transcripts were expressed in IAS muscle. Conclusion Sildenafil relaxes the augmented tone of human IAS in vitro. These results support the potential use of this PDE5 inhibitor in the treatment of chronic anal fissure.