In a previous publication we observed aberrant levels of the human reduced folate carrier (hRFC) in cortex from fetal Down syndrome (DS) subjects. Immunoreactivity for hRFC was increased as the only chromosome 21 gene product studied. We, therefore, analyzed mice transgenic for hRFC (TghRFC1) and wild-type (WT) mice for cognitive functions, behavior and in an observational neurological battery (FOB). Cognitive functions were evaluated by the Morris water maze (MWM), the open field (OF) was used for exploratory behavior, locomotor activity and anxiety-related behavior. The elevated plus maze (EPM) was used to confirm findings in the OF testing anxiety-related behavior and the rota rod (RR) to evaluate motor function. In the MWM TghRFC1 mice performed significantly worse (P < 0.0003) on the probe trial than WT mice. In the FOB visual placing was significantly reduced inTghRFC1 mice. In the OF TghRFC1 mice crossed twice as often (P < 0.029) and in the EPM individuals from this group showed a reduced number of exits from the closed arm (P < 0.044) compared to WT mice. TghRFC1 mice showed impaired performance on the RR, spending one-fourth of the time of WT on the revolving rod (P < 0.0003). Cognitive impairment is an obligatory symptom of DS and this deficiency corresponds to findings in the MWM of mice transgenic for hRFC. Findings of visual placing and failure on the RR may reflect impaired motor performance including muscular hypotonia in DS subjects. Increased crossings in the OF may indicate modulated anxiety-related behavior observed in patients with DS.