The present study was performed as part of a systematic examination of the transmitter specificity of neuronal populations in the larval tiger salamander retina. Backfill-labeling of ganglion cells from the optic tectum was combined with double-label immunofluorescence histochemistry to determine if substance P and GABA are localized to ganglion cell populations in the tiger salamander retina. The triple-label analysis revealed the presence of substance P- and GABA-ganglion cells in both central and peripheral regions of the retina. Substance P-immunoreactive ganglion cells comprised 2% of the total population of backfill-labeled ganglion cells, while less than 1% of backfill-labeled ganglion cells expressed GABA immunoreactivity. Ganglion cells were not found to co-label for both substance P and GABA. Backfill-labeled displaced ganglion cells, which comprised 1.4% of the ganglion cell population, were not observed to be immunoreactive for either substance P or GABA. Forty-six point nine percent of substance P-cells in the ganglion cell layer were backfill-labeled and were identified as ganglion cells. GABA ganglion cells comprised less than 1% of GABA-immunoreactive cells in the ganglion cell layer. Therefore, the present study provides evidence for the presence of small populations of substance P- and GABA-ganglion cells in the larval tiger salamander retina. These observations suggest a functional diversity in the population of tiger salamander ganglion cells relative to their unique transmitter specificities.