Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIalpha and the catalytic subunit Calpha. Low levels of PKA type II consisting of RIIalpha or RIIbeta associated with Calpha were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIalpha and RIIalpha expressions remained in the cytoplasm, while we observed a strong upregulation of RIIbeta, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cbeta splice variants that together with RIIbeta form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function.