Horseradish peroxidase crystals or HRP-NP40 detergent chips were directly applied to brain slices from mice to label primary afferent fibers and their terminal arbors in the brainstem trigeminal complex, and neurons of the ventrobasal complex of the thalamus, their axons in the internal capsule, and their terminals in the primary somatosensory cortex. Anterograde and retrograde labeling of fibers, as well as retrograde labeling of somata, were observed. In vitro labeling of selected non-trigeminal structures and fiber pathways was also demonstrated. Experimental variables have been dealt with in some detail, as have specific advantages and disadvantages of the technique. This in vitro HRP labeling method for neuronal fiber systems is a useful adjunct to currently employed in vivo labeling techniques in the mammalian central nervous system.