Replacement of extracellular chloride ions ([Cl −] o) by other anions, on contractility and the effects of extracellular magnesium ions ([Mg 2+] o]) on spontaneous mechanical activity, as well as on agonist-induced responses of rat aorta ana portal vein, were studied. Replacement of [Cl −] o With acetate (Ac −) or isethionate (Ise −) ions resulted in an increase and decrease, respectively, of the spontaneous mechanical activity frequency in portal vein; the amplitudes of the spontaneous mechanical activity were attenuated by Ac − and Ise − substitution. Withdrawal of [Mg 2+] o in Cl −-containing media resulted in elevation of tension development in rat aortas, whereas a similar maneuver in media with Ac − or Ise −, substituted for [Cl −] o, resulted in abrogation of this tension development. Use of disulfonic stilbene anion-channel blockers, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate, 400–600 μM) and SITS (4,4'-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid, 400–600 μM), failed to influence either spontaneous mechanical activity or basal tone of rat portal portal vein or aortas. Incubation of DIDS or SITS in Mg 2+-free media also failed to influence mechanical responses to withdrawal of [Mg 2+] o. Use of the Cl − cation transport inhibitor bumetanide (30–80 μM) also failed to alter spontaneous mechanical activity or basal tone in either the presence or absence of [Mg 2+] o. Ac − and Ise − substitution attenuated norepinephrine- and K +-induced contractile responses in portal vein and aorta, Caffeine-induced contractions of aortas were potentiated by withdrawal of [Mg 2+] o, in Cl −-containing media but inhibited in Ac −- or Ise − substituted solutions. In the presence of [Mg 2+] o, substitution of foreign anions resulted in alterations in the agonist contractile dose-response curves; EC 50s were increased whereas maximum tensions were depressed. Withdrawal of [Mg 2+] o amplified these effects. Substitution of Ac − or Ise −for [Cl −] o, in the presence or absence of [Mg 2+] o depressed contractions induced by Ca acetate in aortas and portal vein. These results suggest that: (1) Cl − plays an important role in regulating spontaneous mechanical activity, basal tone, and contractility in rat aorta and portal vein; and (2) Cl − probably physiologically mediates some of the effects and actions of [Mg 2+] o. on intracellular release of and influx of Ca 2+ in these smooth muscles.