Modulation of myometrial spontaneity by lead acetate trihydrate (Pb) and its regulatory pathways were studied in estrogenized rats. Isometric tension in myometrial strips under a resting tension of 1 g was measured using data acquisition system-based physiograph and Lab Chart Pro v7.3.7 software. Lead produced a dose-dependent inhibitory effect on rat myometrium with a major effect on phasic contractions compared to tonic contractions along with a reduction in both amplitude and frequency of contraction. Lead (3 μM) significantly (p < 0.05) reduced CaCl2, and 80 mM KDS induced contractile response while potentiated the relaxant effect of phenylephrine. Based on our findings, it may be inferred that lead blocks calcium entry through VDCC and/or stimulates β-adrenoceptors adenylyl cyclase-C-AMP pathway to produce inhibitory effect on rat myometrium.