Diarrhea is caused by factors that alter absorption and secretion of water and ions across the intestinal epithelium and disrupt motility. Parasitic infection, stress, poor nutrition, and exposure to plant or fungal toxins predispose livestock to noninfectious diarrhea. This is more prevalent in sheep that graze pastures infected with wild-type endophytic fungus, suggesting the involvement of fungal alkaloids. These increase smooth muscle contraction: ergovaline/ergotamine (ergot alkaloid) activates serotonin (5-HT) receptors, and lolitrem B (indole diterpene) inhibits large-conductance Ca2+-activated K+ (BK) channels. Because of their separate mechanisms of action the objective of this study was to investigate whether they act synergistically to increase smooth muscle contraction. Effects of ergotamine (1 µM) and lolitrem B (0.1 µM) on the tension and frequency of spontaneous contractions were investigated in a longitudinal preparation of isolated distal colon. The compounds were dissolved in 0.1% dimethyl sulfoxide (DMSO) and applied separately or together for 1 h. Ergotamine increased contractile tension compared to the pretreatment control (P<0.01) and produced a short-lived increase in frequency (P<0.001). Lolitrem B increased contractile tension (P<0.05) but had no effect on frequency. When applied together, the contractile tension was greater than the sum of the compounds applied separately (P<0.05). The frequency of contractions was increased (P<0.05) but was not significantly different from that for ergotamine alone. The increased contractile tension when both compounds were applied together indicates that ergotamine and lolitrem B acted synergistically to increase smooth muscle contraction, suggesting that they would alter motility in vivo.