Behavioral effects of organophosphates (OPs) typically decrease with repeated exposure, despite persistence of OP-induced inhibition of acetylcholinesterase (AChE) and downregulation of muscarinic acetylcholine (ACh) receptors. To characterize this tolerance phenomenon, rats were trained to perform an appetitive operant task which allowed daily quantification of working memory (accuracy of delayed matching-to-position), reference memory (accuracy of visual discrimination) and motor function (choice response latencies and inter-response times during delay). Daily s.c. injections of 0.2 mg/kg of diisopropylfluorophosphate (DFP) caused no visible cholinergic signs, did not affect body weight or visual discrimination, but progressively impaired matching accuracy and lengthened response latencies and interresponse times. These effects recovered in seven of eight treated rats after termination of DFP treatment. Resumption of daily DFP at 0.1 mg/kg caused smaller impairments of both matching accuracy and response latency. After 21 injections of 0.2 mg/kg/day of DFP, rats were subsensitive to the hypothermia induced by acute oxotremorine (0.2 mg/kg i.p.), as expected after OP-induced downregulation of muscarinic ACh receptors. Evidence for supersensitivity to scopolamine (0.03 and 0.056 mg/kg i.p.) in DFP-treated rats was mixed, with additive effects predominating on both the cognitive and motor aspects of the task. After 18 days of 0.1 mg/kg of DFP, AChE was inhibited 50 to 75% and muscarinic ACh receptor density was reduced 15 to 20% in hippocampus and frontal cortex. Progressive declines in AChE activity in hippocampus and frontal cortex across 15 daily doses with DFP at 0.1 and 0.2 mg/kg were observed in other rats; quinuclidinyl benzilate binding was significantly reduced in hippocampus after 15 doses at both levels of DFP. These results indicate that animals showing a definitive sign of tolerance to OP administration (subsensitivity to a cholinergic agonist) were also functionally impaired on both the mnemonic and motoric demands of a working memory task. The nature of this impairment suggests further that it results from compensatory changes in the central nervous system, e.g., muscarinic receptor downregulation, considered to produce "tolerance" to OPs in exposed animals.