Delayed neurotoxicity after acute administration of organophosphates tohenscan be predicted by using the neurotoxic esterase (NTE) assay. The present study was designed to compare results obtained with the assay after single or multiple diisopropyl fluorophosphate (DFP) injections in hens. Adult White Leghorn hens were given DFP in a single (1.0 mg/kg) or in multiple (0.05 mg/kg‐d for 20 d) sc injections. Walking behavior was measured and both groups showed significant impairment by d 5 after the first or only DFP injection. However, impairmen t at the end of the study was greater in hens given a single injection of 1.0 mg/kg. All DFP‐treated hens exhibited axonal degeneration in brains, spinal cords, and peripheral nerves, and the changes in the two groups were of equal severity. Cervical and thoracic spinal cord lesions in hens given multiple DFP injections appeared to be primarily restricted to the myelinated spinocerebellar tracts. Inhibition of NTE was measured in brains, spinal cords, ilea, and thymuses 24 h after the single injection of 1.0 mg/kg DFP or after the 20th injection of 0.05 mg/kg DFP in additional hens. The NTE inhibition was significantly greater in all four tissues in the group given DFP as a single treatment. NTE in nerve andnonnerve tissues appeared to be differentially affected by single and multiple DFP treatments. Walking impairment and NTE inhibition were more marked when DFP was given in a single rather than 20 daily injections. However, in hens given multiple DFP injections the severity of central and peripheral nerve lesions was greater than was expected from the clinical and biochemical results.