We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca²⁺ current (I_Ca) and [Ca²⁺]_i transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation following G_s activation by ,-tubulin. Colchicine depolymerizes microtubules into ,-tubulin dimers. This work analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca²⁺ indicator Fluo-3 and field-stimulated at 1Hz or voltage clamped. In untreated cells, carbachol (CCh, 1µM) induced I_K(ACh), which happens via subunits from the activation of G_i. Carbachol also reduced [Ca²⁺]_i transients and contractions. Once G_i is activated by muscarinic agonist, the _i subunit is released from the but it is silent and its inhibition of the AC/cAMP cascade, manifested by I_Ca reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca²⁺]_i transients and contractions than in untreated cells. The _i subunit became effective in signaling through the AC/cAMP cascade and reduced I_Ca without changing its voltage-dependence. ISO regained its efficacy and reversed I_Ca inhibition by CCh. Stimulation of I_Ca by forskolin persisted in colchicine-treated cells when ISO was ineffective. The effect of CCh on I_K(ACh) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase I_K(ACh) by subunits released from G_s to mask this effect of CCh. Microtubules suppress I_Ca regulation by _i; their disruption releases restraints that unmask muscarinic inhibition of I_Ca. Summarily, colchicine treatment reverses the regulation of ventricular excitation-contraction coupling by autonomic agents.