A fluorescent molecular probe was used in combination with a novel in vitro preparation to study spatial patterns of neural activity associated with motor pattern generation. The in vitro brainstem- cerebellum preparation takes advantage of the turtles's unusual resistance to anoxia to preserve the entire neural network that connects the cerebellum, red nucleus, and reticular formation. This preparation was bathed in a 0.01% solution of sulforhodamine while it was activated unilaterally by electrical stimulation of the dorsal quadrant of the spinal cord for 1 hr. Sulforhodamine is a small, sulfonated, highly charged fluorescent molecule that is taken up by endocytosis. To examine its distribution in the cerebellum and brainstem, coronal sections were prepared and viewed under epifluorescence illumination. Distinctive spatial patterns of labeling were associated with unilateral electrical stimulation of the in vitro network, suggesting that dye uptake was activity dependent. Blockade of uptake with altered magnesium and calcium concentrations indicated that single spike discharge evoked ortho- or antidromically was insufficient to induce dye uptake. Instead, sulforhodamine staining correlated with the presence of burst discharge that was recorded extracellularly from the red nucleus. Blockade of burst discharge with excitatory amino acid receptor antagonists prevented dye uptake in the red nucleus, the lateral cerebellar nucleus, and other structures that are known to be interconnected by recurrent anatomical pathways. These results suggest that sulforhodamine is internalized by intensely active neurons. The spatial distributions of label support the hypothesis that burst discharges in the turtle red nucleus are mediated by excitatory amino acid neurotransmitters and sustained by recurrent excitation in cerebellorubral synaptic pathways. Positive feedback in these recurrent pathways may provide an important driving force for the generation of motor programs that control limb movements.