The anatomical source of the trail pheromone in the black carpenter ant, Camponotus pennsylvanicus (De Geer), was investigated by presenting workers with hindgut extracts, testing for attractancy and trail-following behavior. Chemical analyses were used in conjunction with behavioral bioassays to detect and identify volatiles from the rectum, poison gland, and Dufour’s gland. Rectal material was also examined to determine total nitrogen levels and identify nitrogenous metabolites. Under laboratory conditions, foragers demonstrated a significant level of attraction to a combined Dufour’s gland, poison gland, and rectal extract, although no trail following was observed. Two volatiles, n-undecane and n-tridecane, were identified from Dufour’s gland. Fatty acids and esters were detected in Dufour’s and poison gland extracts. Palmitic acid was identified in the poison gland. A compound described as a component of the trail pheromone in Camponotus atriceps Smith (3,4-dihydro-3,5-dihydroxy-6-methyl-pyran-4-one) was identified in the rectum, but was not verified behaviorally as being part of the trail pheromone for C. pennsylvanicus. Dry weight analysis showed that the rectal material was only 14% solid material and total nitrogen levels were estimated at 19.2 ± 2 μg/mg. Most of the components contributing to the total nitrogen excreted were unidentified, but ammonia (2.7 ± 1.2 μg/mg), two tryptophan intermediates (kynurenic and xanthurenic acid) and one pteridine (biopterin), were identified.