Working memory in rats involves neural projections from the hippocampus (HP) to the prefrontal cortex (PFC), based on delayed task experiments in a radial-arm maze, in which the time span of working memory is longer than seconds. To determine whether the HP–PFC pathway is involved in short-term (on the order of seconds) working memory function, we lesioned the PFC and/or HP, and measured performance in an operant delayed alternation task. The posterior dorsal (pdHP) and ventral HP (vHP) were assessed separately. The bilateral PFC and bilateral pdHP ibotenate lesions produced significant working memory deficits, but the vHP lesion did not. Unilateral pdHP lesions combined with a PFC lesion in the opposite hemisphere reproduced the effects of bilaterally symmetrical lesions. By contrast, unilateral lesions of the pdHP combined with a PFC lesion in the same hemisphere had no effect on delayed alternation. These results indicate that the pdHP–PFC pathway is essential for working memory on the order of seconds in rats, and suggest that the pdHP and vHP pathways to the PFC play different behavioral roles.