Spatial memory has been strongly associated with hippocampal function. There are several reports of the participation of this structure in acquisition and consolidation of spatial tasks. In this study, we evaluated the effects of selective and non-selective muscarinic antagonists in the dorsal hippocampus of rats during acquisition and encoding of a spatial task. Rats were trained in a Morris water maze for 4 days with identical daily sessions, and tested for long-term memory (LTM) 1 week after training. The animals were injected bilaterally in the dorsal hippocampus 20 min before the start of every day of training. The results showed that the non-selective muscarinic antagonist, scopolamine, disrupted acquisition of water maze memory formation. Moreover, microinjections of a selective postsynaptic muscarinic antagonist, pirenzepine, disrupted LTM, whereas it did not affect acquisition. Conversely, a selective presynaptic muscarinic antagonist, AFDX-116, did not disrupt either water maze acquisition or LTM formation. Combination of AFDX-116 and pirenzepine had similar effects as scopolamine, partially blocking acquisition and impairing long-term spatial memory. These results support the view that muscarinic receptors are involved in spatial learning and that postsynaptic muscarinic receptors in the dorsal hippocampus are particularly involved in long-term spatial memory formation.