The use of retrograde axonal transport of various substances (for example, enzymes, lectins, synthetic fluorescent compounds) has yielded much information on the organization of neuronal pathways. Each type of retrograde tracer has its own set of attributes which define the scope of problems it can address. We describe here a new class of retrograde tracer, rhodamine-labelled fluorescent latex microspheres (0.02-0.2 micron diameter), which have distinct advantages over other available tracers for in vivo and in vitro applications. When injected into brain tissue, these microspheres show little diffusion and consequently produce small, sharply defined injection sites. Once transported back to neuronal somata, the label persists for at least 10 weeks in vivo and 1 yr after fixation. Microspheres have no obvious cytotoxicity or phototoxicity as assessed by intracellular recording and staining of retrogradely labelled cells in a cortical brain slice preparation. This approach was further used to visualize and compare, in cat visual cortex slices, neurones with different projection patterns, and revealed significant differences in patterns of intrinsic axons and dendrites. These properties of microspheres open new avenues for anatomical and physiological studies of identified projection neurones in slices as well as in dissociated cell cultures.