For a successful human pregnancy, the phasic smooth muscle of the myometrium must remain quiescent until labor. Activation of cAMP/cAMP-dependent protein kinase A (PKA) pathways contributes to this quiescence. The small heat-shock protein 20 (HSP20) is a target of PKA, and phosphorylated HSP20 (pHSP20) modulates relaxation of tonic vascular smooth muscle via interaction with actin, independent of myosin dephosphorylation. Our objective was to determine whether relaxation in human myometrium is associated with changes in phosphorylation of HSP20. Myometrium was obtained at elective cesarean. Elevating cAMP with forskolin or rolipram (a phosphodiesterase inhibitor) caused substantial relaxation of spontaneously contracting human myometrial strips, of 92 +/- 4% (mean +/- sem, n = 10) and 84 +/- 7% (n = 6), respectively. Subsequent two-dimensional electrophoresis with immunoblotting of strip extracts showed a significant 2.6- and 2.1-fold increase in phosphorylated HSP20 (pHSP20) after forskolin (P < 0.01; n = 5) or rolipram treatment (P < 0.05; n = 4). Noncyclic-nucleotide-mediated relaxation, induced by the calcium channel blocker nifedipine, did not alter pHSP20. Inhibition of PKA with H89 significantly attenuated rolipram-induced relaxation (P < 0.01; n = 4), and partially reduced rolipram-stimulated pHSP20. Total and pHSP20 protein was unchanged in term laboring and nonlaboring myometria. Coimmunoprecipitation studies revealed a specific association of HSP20 with alpha-smooth muscle actin and HSP27, a key regulator of actin filament dynamics. Finally, coimmunofluorescence demonstrated moderate colocalization of HSP20 with alpha-smooth muscle actin in the cytoplasm of laboring myometria. Our data support a novel role for pHSP20 in the modulation of cyclic-nucleotide-mediated myometrial relaxation, through interaction with actin. pHSP20 represents an important new target for future tocolytic therapy.