Smooth muscle cells isolated from the myometrium of a pregnant woman at term were infected with a replication-defective adenovirus vector expressing the E6/E7 proteins of human papilloma virus 16. A clonal line, PHM1-41, was selected by resistance to Geneticin and examined for maintenance of smooth muscle phenotype and response to oxytocin. The immortalized cell line retained morphological characteristics of proliferating smooth muscle cells in culture for up to 22 passages and has been used for over 2 years. The cells expressed smooth muscle-specific alpha-actin and retained estrogen receptors. Oxytocin receptors were present, as measured by whole cell binding assay using the oxytocin antagonist 125I-d(CH2)5[Tyr-(Me)2,Thr4,-Orn8,Tyr9-NH2] as ligand and oxytocin as competitor. The data were best described by a one-site binding model, with a Kd of 0.36 nM and a binding site concentration of 37 fmol/microgram DNA. PHM1-41 cells responded to oxytocin with an increase in intracellular free calcium (EC50 15 nM) and an increase in phosphatidylinositol turnover. Oxytocin-stimulated phosphatidylinositol turnover was inhibited by preincubation with the cAMP analog CPT-cAMP. This immortalized myometrial cell line should prove useful for studies relating to human myometrial function.