Attentional functioning in mice was assessed in an analogue of the five-choice serial reaction time task in which the requirement was to detect brief visual stimuli presented across five spatial locations. Two hybrid strains of mice were assessed; F1 C57Bl/6xDBA/2 and C57Bl/6x129sv. Both strains acquired the task to high levels of performance with, in particular, no problems due to premature responding. At performance, systematic manipulation of the task parameters indicated a pattern of effects consistent with the task, taxing aspects of visuospatial attention. There were some differential effects of task manipulations at baseline across strain. However, the pattern of effects suggested these were likely to be the result of effects on factors other than attentional functioning per se, such as behavioural reactivity and inhibition. There was evidence in both strains of specific, centrally mediated effects of scopolamine on attentional functioning, with the C57Bl/6xDBA/2 hybrid showing greater sensitivity to the drug manipulation. Specific effects on discriminative accuracy were observed at doses of 0.02 and 0.2 mg/kg scopolamine. At the 2 mg/kg dose, large reductions in accuracy were associated with large effects on other measures, including omissions and response latencies, suggestive of nonspecific effects on task performance. These data indicate, for the first time, the utility of operant methods in assessing visuospatial attentional functioning in mice. They confirm the importance of cholinergic mechanisms in attentional processes across species, and suggest interactions between cholinergic mechanisms and genotype in the expression of attentional phenotypes.