We addressed the question whether the projection neurons of the olfactory bulb, i.e. the mitral and tufted cells, are immunoreactive for the calcium-binding protein, calretinin. The following approaches were adopted: (1) light and electron microscopic calretinin-immunocytochemistry; (2) neuroanatomical tracing combined with calretinin-immunocytochemistry according to double-peroxidase and double-fluorescence protocols; (3) unilateral lesion of the olfactory bulb combined with calretinin-immunocytochemistry. The experiments were carried out in rats. Immunostaining of brain sections revealed weakly calretinin-immunopositive mitral cell bodies. Tufted cells were immunonegative. In contrast, fibers in the lateral olfactory tract were strongly immunopositive. Dense immunostaining was also present in a superficial band in layer I of the olfactory tubercle, piriform cortex, periamygdaloid cortex, and in the lateral entorhinal cortex. In electron microscopic preparations of these target areas we observed immunoreaction product in axons and axon terminals. The latter invariably formed asymmetrical synapses, mostly with dendritic spines. Injections of the neuroanatomical tracer biotinylated dextran amine (BDA) into the olfactory bulb produced labeled fibers which remained completely restricted to the superficial, calretinin-immunopositive band in layer I in the above-mentioned cortical forebrain areas. We noted colocalization of transported BDA and calretinin-immunoreactivity in mitral cells, in fibers in the lateral olfactory tract and in fibers in the piriform cortex. Olfactory bulb lesions produced depletion of calretinin-immunoreactivity in the lateral olfactory tract and the superficial band in the olfactory cortex-related areas. Together these data firmly indicate that mitral cells and their axons are calretinin-immunoreactive.(ABSTRACT TRUNCATED AT 250 WORDS)