The clinical effects of two neurotoxicants, acrylamide and 2,5-hexanedione, were compared in rats using a functional observational battery (FOB), which includes a series of home cage and open-field observations, sensorimotor measurements, and physiological parameters. Neurotoxicity was assessed weekly in adult male Long-Evans rats after initiation of IP administration of 9 doses of acrylamide (12, 15, or 50 mg/kg given 3 times a week) and 28 doses of 2,5-hexanedione (150, 225, and 350 mg/kg given daily). Using the FOB, it was possible to detect differences in neurotoxic effects of these two chemicals. Acrylamide significantly affected home cage posture, foot splay and time on the rotarod, whereas 2,5-hexanedione altered hindlimb grip strength and the approach response. Both compounds caused changes in ability to walk, right, and maintain agility on a rotarod within 21 days from initiation of toxicant administration. In addition, both compounds caused dose-dependent decreases in weight gain. Neuropathic changes were detectable at the highest dosages at 21 days in acrylamide-treated rats and at 28 days in rats treated with 2,5-hexanedione. Administration of acrylamide also decreased activities of neural esterases. This study indicated that the FOB could be used to detect evidence of neurotoxicity in rats treated with acrylamide and 2,5-hexanedione, with alterations evident even before pathological changes were induced by 2,5-hexanedione.