The N-methyl-D-aspartate R1 (NMDA R1), NMDA R2A, and NMDA R2C subunits were expressed transiently in double or triple combinations in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult mouse forebrain and cerebellum. Under conditions established for maximal expression, cotransfection of the NMDA R1 and R2C subunits yielded a protein detected immunologically with a molecular size of 780,000-850,000 daltons. No cell death was observed in the transfected cells, and the KD for [3H]MK801 binding to the NMDA R1/R2C receptor was 346 +/- 158 nM. This was in contrast to a value of KD = 22 +/- 9 nM found for native cerebellar receptors. Co-transfection with NMDA R1/R2A/R2C subunits with a DNA ratio, 1:3:3, resulted in the expression of a protein with a size similar to the NMDA R1/R2C combination, but the affinity of [3H]MK801 was now 22 +/- 5 nM, and the percentage cell death post-transfection was 89 +/- 17%. Immunoprecipitation assays of detergent-solubilized transfected cells with NMDA R1 subunit-specific antibodies co-precipitated the NMDA R2A and NMDA R2C subunits in 1/2A and 1/2C transfections, respectively. Similarly, immunoprecipitations with either NMDA R1 or NMDA R2C subunit-specific antibodies co-precipitated the NMDA R2A subunit in the R1/2A/2C triple transfections. These results show that the three NMDA receptor subunit types can co-assemble following their co-expression in mammalian cells with a pharmacological profile that is similar to that found for adult cerebellar NMDA receptors.