BACKGROUND: Symptomatology and physiology differ between men and women and across the menstrual cycle in irritable bowel syndrome (IBS). Ovarian hormones influence 5-hydroxytryptamine (5-HT), an amine known to play a role in gut motor-sensory function. AIM: To assess the effects of gender and menstrual status on platelet-depleted plasma (PDP) 5-HT concentration in IBS patients with diarrhoea (IBS-D) patients compared with healthy volunteers (HV). METHODS: Platelet-depleted plasma 5-HT concentrations were assessed under fasting and fed conditions in 73 IBS-D patients (aged 18-58 years; 18 men) and 64 HV (aged 18-50 years; 24 men). Women were divided into those with low or high progesterone/oestrogen (P/O) levels. RESULTS: Irritable bowel syndrome patients with diarrhoea had higher PDP 5-HT concentrations than HV under fasting (P = 0.002) and fed (P = 0.049) conditions. This was particularly related to IBS-D men having higher PDP 5-HT concentrations than healthy controls (P = 0.002). Moreover, PDP 5-HT concentrations in IBS-D women with low P/O levels were similar to healthy controls. CONCLUSIONS: Similar to IBS-D women with high P/O levels, IBS-D men also have raised PDP 5-HT concentrations. 5-HT concentration normalizes at menses in IBS-D women, suggesting a shift in the mechanisms responsible for abnormal 5-HT signalling in these patients.