Cholinergic blockade has been shown to impair performance in delayed nonmatching to position (DNMTP) paradigms in rats. In this study, a murine operant DNMTP task was used to assess the effects of cholinergic antagonism in two strains of mice (DBA/2 and C57BL/6) differing in spatial learning abilities. DNMTP was scheduled in operant chambers with retractable levers, where mice were trained until high levels of accuracy. Subsequently, proactive interference effects were assessed by manipulation of the intertrial interval (ITI), and animals were tested in this task under scopolamine (0.1-1.0 mg/kg) and mecamylamine (0.5-2.0 mg/kg) treatment. Data were analyzed according to the methods of signal detection theory. ITI manipulation decreased accuracy when the time between trials was reduced to 5 s. Cholinergic blockade failed to induce a pure mnemonic impairment but distinguishable effects of both receptor antagonists could be detected: scopolamine disrupted accuracy in a dose-dependent but delay-independent manner, whereas mecamylamine failed to impair accuracy, but decreased responsivity delay- and dose-dependently. Strains mainly differed in responsivity, with DBA/2 showing higher latencies to respond to the levers. These results are comparable to those obtained in rats. Thus, operant DNMTP can be applied to assess working memory in mice.