Substance use disorders are characterized by excessive seeking and taking of drugs in the face of adverse consequences. Impaired learning of contingencies around negative outcomes may influence such drug-use patterns. To our knowledge, no studies to date have examined fear conditioning in drug-dependent humans, nor have they investigated the association between fear conditioning outcomes and neuropsychological functioning. The current study compared de novo fear conditioning in a sample of opiate-dependent outpatients to that in a healthy and an affective-disordered sample in an attempt to isolate the contribution of substance dependence on conditioning outcomes, relative to the substantial comorbidity present in substance using samples. Additionally, a brief neuropsychological battery was completed in the substance dependent sample. We found consistent evidence across three indices of fear acquisition—simple conditioning, differential conditioning, and contingency awareness—that the opiate-dependent learned the cue association less well than the control groups. Some of these deficits were significantly related to impairments in attention and memory. As such, substance use and/or associated neuropsychological impairments may make associative learning difficult, and may contribute to the persistence of maladaptive patterns and the failure to change behavior in response to aversive outcomes.