A polyclonal antiserum to a fusion protein corresponding to a region of the NMDA R1 (NR1) subunit (amino acids 656-811) was produced and affinity purified. A quantitative immunoblotting technique was developed using the fusion protein as a standard. By employing this method, ontogenic studies (day 2-42) of the density of NR1 protein were carried out in several regions of rat brain. The results showed that in all five of the brain regions examined [olfactory bulb (Ob), cortex (Cx), hippocampus (Hp), midbrain (Mb) and cerebellum (Cb)], levels of NR1 protein are low at birth and increase with similar patterns having a sharp rise within the first 3 weeks after birth. Levels increased 2.0 to 4.5-fold from postnatal day 2 to postnatal day 42. Although the general patterns of developmental expression are similar, large differences in the absolute amounts of NR1 protein among the five brain regions were observed. The maximal levels (pmol of fusion protein equivalent/mg +/- S.E.) of NR1 subunit attained during development in the five regions are: Hp 2.0 +/- 0.37 > Cx 1.4 +/- 0.11 > Ob 1.3 +/- 0.2 > Mb 1.0 +/- 0.10 > Cb 0.57 +/- 0.13. The temporal patterns of expression of NR1 protein are similar to results from studies examining the expression of NR1 mRNA. Furthermore, the absolute numbers obtained from our studies are close to those found using [(3)H]MK-801 binding suggesting that many of the NR1 subunits expressed in the brain exist in an active form.