We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K(d), 10 nm) for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTPgammaS and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues. Neurobeachin, BGL, and approximately 10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat protein-like membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates.