We studied the mechanisms underlying the increase in automaticity induced by α1-adrenergic stimulation of normal and “ischemic” canine Purkinje fibers. Fibers were superfused with a control Tyrode's solution, followed by an ischemic superfusate that included 10 mM KCI,5 mM NaHCO3, Po2of 10–25 mm Hg, and pH 6.7. To exclude β-adrenergic actions, propranolol was added to all solutions. In the presence of phenylephrine, normal automaticity at high membrane potentials usually decreased, whereas the incidence of abnormal automaticity during ischemia was increased from a control value of 10% to 30%. Block of an α1-receptor subtype with chloroethylclonidine in the presence of phenylephrine caused normal automaticity to increase in all fibers studied and significantly increased abnormal automaticity to 70%. The α-adrenergic-induced increase in automaticity did not occur in ischemic fibers from animals pretreated with pertussis toxin (PTX), which ADP-ribosylated and functionally inactivated the 41-kd family of GTP regulatory proteins. In contrast, the use of PTX enhanced the increase in automaticity induced by phenylephrine in normally polarized Purkinje fibers. Ryanodine, which blocks sarcoplasmic reticulum Ca2+release, attenuated the increase in normal automaticity in nonischemic fibers but had no effect on abnormal automaticity in ischemic fibers. The increase in abnormal automaticity was, however, blocked by the α1subtype blocker WB 4101, which also blocks the increase in automaticity in normal fibers. In conclusion, the increase in abnormal automaticity in ischemic Purkinje fibers depends on a WB 4101-sensitive α1-adrenergic receptor subtype whose actions are transduced by a PTX-sensitive 41-kd G protein and are not blocked by ryanodine. This is clearly different from the mechanism underlying the increase in automaticity in normal Purkinje fibers, which is independent of the PTX substrate but is suppressed by ryanodine.