5-Hydroxytryptamine (5-HT) receptors mediating excitatory and inhibitory actions of 5-HT on contractility of uterine strips from non-pregnant pigs were characterized. Expression of 5-HT2A and 5-HT7 receptors was examined by molecular biological study. 5-HT-containing cells were observed immunohistochemically. In the spontaneously contracting uterine circular muscle layers, 5-HT caused inhibition of contractile activity. SB269970 (5-HT7 receptor antagonist, 10 nM) shifted the concentration-inhibition curve of 5-HT to the right, but higher concentrations of SB269970 (100 nM–1 μM) changed the monophasic curve to a biphasic curve (mixture of excitatory and inhibitory responses to 5-HT). Addition of ketanserin (5-HT2 receptor antagonist, 10 nM–1 μM) decreased the excitatory effects of 5-HT. 5-HT was less effective in inhibiting the spontaneous contraction in the longitudinal muscles. Ketanserin enhanced the inhibitory responses and SB269970 reversed the inhibitory responses to excitatory responses. In the presence of SB269970, α-methyl-5-HT was equipotent to 5-HT in increasing contractility of longitudinal muscle and ketanserin competitively inhibited the responses to α-methyl-5-HT (pKd = 8.78). Muscle layer-dependent expression of both 5-HT2A receptor and 5-HT7 receptor mRNAs in the porcine uterine muscle layers was demonstrated by RT-PCR and real-time PCR. 5-HT immunoreactivity was detected only in uterine gland cells, which were localized near the uterine circular muscle layers. In the longitudinal and circular muscle layers with endometrium, compounds 48/80 and ketanserin did not change the spontaneous contractility, but SB269970 significantly increased the contractile activity of the circular muscle. In conclusion, excitatory 5-HT2A and inhibitory 5-HT7 are present in the uterus of non-pregnant pigs. Endogenous 5-HT containing cells are mainly present in uterine glands of the pig. The possible roles of 5-HT and its receptors in regulation of porcine uterine spontaneous contractility are discussed.