We investigated the role of protein kinase C (PKC) in ₁-adrenergic regulation of intracellular Na⁺ activity (a_Naⁱ) in single guinea pig ventricular myocytes. a_Naⁱ and membrane potentials were measured with the Na⁺-sensitive indicator sodium-binding benzofuran isophthalate and conventional microelectrodes, respectively, at room temperature (24-26°C) while myocytes were stimulated at a rate of 0.25-0.3 Hz. The PKC activator 4-phorbol 12-myristate 13-acetate (PMA) decreased a_Naⁱ in a concentration-dependent manner. PMA (100 nM) produced a maximal decrease in a_Naⁱ of 1.5 mM from 6.5 ± 0.4 to 5.0 ± 0.4 mM (means ± SE, n = 12, P < 0.01). The PMA concentration required for a half-maximal decrease in a_Naⁱ was 0.46 ± 0.13 nM (n = 3, P < 0.01). An inactive phorbol, 4-phorbol 12-myristate 13-acetate, did not decrease a_Naⁱ. The decrease caused by PMA could be blocked by the PKC inhibitors staurosporine and bisindolylmaleimide I (GF-109203X). Stimulation of the ₁-adrenoceptor with 50 µM phenylephrine decreased a_Naⁱ from 6.1 ± 0.3 to 4.6 ± 0.3 mM (n = 11, P < 0.01). The decrease in a_Naⁱ produced by phenylephrine was blocked by pretreatment with staurosporine, GF-109203X, or PMA. The decrease in a_Naⁱ produced by PMA was not prevented by pretreatment with tetrodotoxin but was blocked by pretreatment with strophanthidin or high extracellular K⁺ concentration. The results suggest that ₁-adrenergic receptor activation results in a decrease in a_Naⁱ via PKC-induced stimulation of the Na⁺-K⁺ pump in cardiac myocytes.