Objective-To determine effects of short-chain fatty acids (SCFA) on canine colonic smooth muscle. Sample Population-Colonic tissue obtained from 14 healthy dogs. Procedure-Short-chain fatty acid (SCFA; acetate, propionate, and butyrate; 1 to 100 mmol/L)-induced contractions were compared with responses obtained with acetylmethylcholine (AMCh,10-4 mol/L). Roles of enteric neurons, cholinergic receptors, calcium stores in the sarcoplasmic reticulum, and extracellular calcium in the SCFA-incluced responses were investigated by incubating muscle strips with tetrodotoxin (1 μmol/L), atropine (1 μmol/L), ryanodine (10 μmol/L), nifedipine (1 μmol/L), ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′-tetra-acetate (EGTA; 0.1 mmol/L), or an extracellular calcium-depleted (zero extracellular calcium) solution prior to the addition of propionate or butyrate. Results-Incubation with SCFA elicited isometric stress responses (0.25 to 2.15 × 104 N/M2) in colonic longitudinal smooth muscle. Maximal responses to butyrate and propionate (50 mmol/L) were 37 and 23%, respectively, of the maximal AMCh response. Acetate was least effective in stimulating contractile responses. Tetrodotoxin and atropine did not affect SCFA-induced contractions. Nifedipine and zero extracellular calcium solution abolished responses to butyrate and propionate, whereas EGTA attenuated (> 60%) but did not abolish those responses. Ryanodine did not affect SCFA-induced contractile responses. The SCFA did not affect colonic circular smooth muscle. Conclusions and Clinical Response-The SCFA stimulate longitudinal but not circular colonic smooth muscle contractions via a direct effect on smooth muscle. The mechanism of the SCFA effect appears to involve the influx of extracellular calcium. These findings may account for some of the effects of canine colonisc motility.