Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.