Two major forms of mammalian acetyl-CoA carboxylase (EC 6.4.1.2), ACC-α and ACC-β, have been described and the sequences of the isoforms deduced. ACC-β is the predominant isoform expressed in heart and skeletal muscles, in which a major role of malonyl-CoA is probably to regulate fatty acid β-oxidation. The regulatory properties of ACC-β are incompletely defined but it is known that some cellular stresses lead to inhibition in parallel with the activation of AMP-activated protein kinase (AMP-PK). Here we examine the phosphorylation state of ACC-β within intact rat cardiac ventricular myocytes. Treatment of myocytes with the β-adrenergic agonist isoprenaline (isoproterenol) led to increased ACC-β phosphorylation that was maximal within 2 min and with 50 nM agonist. Effects of isoprenaline were revealed by the incorporation of 32P into ACC in cells incubated with [32P]Pi and also by a marked decrease (approx. 80%) in subsequent phosphorylation in vitro with cAMP-dependent protein kinase (PKA). Analysis of tryptic phosphopeptides revealed that ACC-β was phosphorylated at multiple sites by incubationin vitro with PKA or AMP-PK. Treatment of myocytes with isoprenaline affected all the major phosphorylation sites of ACC-β that were recognized in vitro by purified PKA, so that subsequent phosphorylation in vitro was greatly diminished after cell stimulation. β-Adrenergic stimulation led to decreases in cellular malonyl-CoA concentrations but no changes in kinetic properties of ACC were detected after cell homogenization and partial purification of proteins. The results suggest that: (1) ACC-β is rapidly phosphorylated at multiple sites within intact cardiac ventricular myocytes after β-adrenergic stimulation, (2) ACC-β is phosphorylated in vitro by PKA and AMP-PK at multiple sites, including at least one site accessible to each kinase, as well as kinase-selective sites, and (3) PKA is a physiologically significant ACC-β kinase.