OBJECTIVE: Functional gastrointestinal disorders affect females more often. Changes in colonic motility may be etiological co-factors for the clinical symptoms. The aim of the present study was to analyze the influence of gonadal hormones on colonic contractile activity. METHODS: In vitro measurements of colonic contractile activity in longitudinal smooth muscle strips of female and male Lewis rats were performed in an organ chamber experiment. After the administration of a gonadal hormone estradiol [EST], progesterone [PROG] and testosterone [TEST]) or ethanol solution as control, stimulation with acetylcholine (ACh) or inhibition with norepinephrine (NE) was performed. RESULTS: Compared to the smooth muscle strips of male rats, significantly higher spontaneous colonic contractile activity (SCCA) was observed in female animals. Increasing doses of ACh showed the progressive stimulation of SCCA whereas rising doses of NE resulted in a stepwise inhibition of SCCA, respectively. EST superfusion displayed an inhibitory effect on SCCA in both sexes and inhibited the ACh effect in female rats. Similarly, acute superfusion with high-dose PROG inhibited SCCA in females. Acute TEST superfusion inhibited SCCA in males and led to significant higher colonic contractile activity in males following subsequent stimulation with ACh. In female rats, the inhibitory effect of NE was reduced by prior exposure to TEST. CONCLUSION: In our in vitro study the acute exposure of colonic smooth muscle tissue to gonadal hormones led to sex-dependent changes in SCCA and translated in a modified response of smooth muscle strips to both pro-contractile and anti-contractile neurotransmitters.