We have documented gestation- and labour- (preterm and term) dependent changes in expression of genes encoding contraction associated proteins in the rat uterus and correlated these changes with various parameters of uterine contractility. The data demonstrate increased expression of contractile agonist systems concurrent with decreased expression of relaxant systems after gestational day 20. Significant increases in expression of oxytocin (OT), its receptor (OTR), prostaglandin (PG) H synthase isoform 1 (PGHS-1) and PGF2α receptor (FP) occurred first, followed by increases in PGHS-2, connexin-43, endothelin-1 (ET-1) and the ET-1 receptor isoform ETA. Expression of OTR and FP was significantly reduced during mid-gestation compared to non-pregnant animals. Expression of inducible nitric oxide synthase (iNOS) increased significantly during pregnancy then decreased concurrently with the increase in OTR and FP. Functional changes in uterine contractility accompany changes in gene expression. OT was the most potent contractile stimulant. Sensitivity of uterine strips to OT was reduced in early and mid-pregnancy then increased at uterine activation. Progesterone antagonist-induced preterm labour caused changes similar to those at normal term. Comparison of mRNA transcripts in separated endometrium and myometrium suggested that the endometrium is an important regulator of myometrial contractility, analogous to the relationship between endothelium and vascular smooth muscle. This novel combination of functional and genetic expression analyses provides new insight into the physiology of parturition.