The effects of soy phytoestrogens on Morris water maze (MWM) performance and neuronal cholinergic enzyme activities and immunoreactivity were studied in ovariectomized (OVX) rats. The rats were assigned to four groups fed control diet (CD), 3.9 mg/kg 17beta-estradiol diet (E2), 263.4 mg/kg soy phytoestrogens diet (SP1), and 526.9 mg/kg soy phytoestrogens diet (SP2). In the MWM task, escape latency and path length were significantly less in the E2 and SP2 groups than in the CD group on the second day. Choline acetyltransferase (ChAT) activity in the cerebral cortex and ChAT immunoreactivity in the diagonal band of Broca were significantly greater in the E2, SP1, and SP2 groups than in the CD group. Acetylcholinesterase activity in the hippocampus in the E2, SP1, and SP2 groups was significantly lower than in the CD group. This study suggests that soy phytoestrogens affect the reference memory and neuronal cholinergic system in OVX rats.