The underlying mechanisms regulating uterine contractions during labour are still poorly understood. Heat shock protein 20 (HSP20) is known to be present at high levels in smooth muscle and implicated in muscle relaxation, but HSP20 expression in the myometrium is completely undetermined. Since HSP20 has been implicated in smooth muscle relaxation, we hypothesized that HSP20 would be highly expressed in the rat myometrium during early and mid-pregnancy when the myometrium is relatively quiescent. Northern blot analysis particularly demonstrated that HSP20 mRNA detection was significantly decreased from day (d) 22 of pregnancy to 1-day post-partum (PP) compared with d6 (P < 0.05). HSP20 mRNA detection was also significantly decreased from d22 to d23 of gestation compared with non-pregnant (NP) samples. Immunoblot analysis showed that detection of HSP20 was significantly decreased at d23 compared with d12 and d15 (P < 0.05). HSP20 detection also significantly decreased at PP compared with d15 (P < 0.05). Immunofluorescence analysis demonstrated that after d15, plasma membrane-associated localization of HSP20 decreased markedly in both circular and longitudinal muscle layers. In addition, HSP20 was detectable near cell membranes at much higher levels in the longitudinal muscle layer of progesterone-treated pregnant rats (delayed labour) at all gestational time points examined, compared with controls. Our results demonstrate that HSP20 mRNA and protein are highly expressed during early and mid-pregnancy and then the expression markedly decreases during late pregnancy and labour. The observed patterns of HSP20 expression are consistent with a potential role for HSP20 in facilitating myometrium quiescence during early and mid-pregnancy.