Folate is essential for fetal development, and its deficiency during gestation causes behavioural deficits in the offspring. The present study investigated its influence during weaning on brain function in the pups of rats that were put on a folate-deficient (FD) diet on postnatal day (PND) 1. Systemic folate deficiency in pups on the FD diet (n 15) was evident from the dramatically lower hepatic folate concentrations (median 23·7, range 8·1–48·4 ng/mg protein) and higher homocysteine concentrations (median 27·7, range 14·7–45·5 pmol/mg protein), respectively, compared with those of pups on the normal diet (ND; n 9) (median 114·5, range 64·5–158·5 ng/mg protein and median 15·5, range 11·6–18·9 pmol/mg protein) on PND 23. Brain folate concentrations although low were similar in pups on the FD diet (median 10·5, range 5·5–24·5 ng/mg protein) and ND (median 11·1, range 7·1–24·2 ng/mg protein). There was a high accumulation of homocysteine in the brain of FD pups, mostly in the hippocampus (median 58·1, range 40·8–99·7 pmol/mg protein) and cerebellum (median 69·1, range 50·8–126·6 pmol/mg protein), indicating metabolic folate deficiency despite normal brain folate concentrations. Developmental deficits or autistic traits were more frequent in the FD group than in the ND group and repetitive self-grooming occurred, on average, three times (range 1–8) v. once (range 0–3) during 5 min, respectively. Long-term memory or spatial learning and set-shifting deficits affected 12 to 62 % of rats in the FD group compared with none in the ND group. Post-weaning folic acid supplementation did not correct these deficits. These observations indicate that folate deficiency during weaning affects postnatal development even when gestational folate supply is normal.