Presynaptic N-methyl-D-aspartate (NMDA) receptors in terminals of primary afferents to spinal cord of rats were first reported by Liu et al. (1994; Proc. Natl. Acad. Sci. USA 91:8383-8387) and were proposed to modulate nociceptive input (Liu et al. [1997] Nature 386:721-724). We previously demonstrated kainate and AMPA receptors in numerous primary afferent terminals in the spinal cord fixed with diluted paraformaldehyde and no glutaraldehyde. Therefore, we reinvestigated the occurrence of presynaptic NMDAR1 (NR1) with this fixation protocol. With confocal microscopy, numerous immunofluorescent puncta were double-stained for NR1 and the presynaptic marker synaptophysin throughout the spinal gray. NR1-immunostained puncta costained more frequently with a tracer that labels myelinated afferents (cholera toxin subunit B; CTB) than with a tracer that labels non-peptidergic unmyelinated afferents (Griffonia simplicifolia isolectin B4; IB4). Virtually no double staining was found for NR1 and calcitonin gene-related peptide (CGRP), which labels somatic peptidergic primary afferents. In the gracile nucleus, virtually all puncta labeled for CTB appeared immunopositive for NR1. At the electron microscopic level, most immunopositive terminals in spinal cord and gracile nucleus displayed morphological characteristics of endings of myelinated primary afferents. NR1 was presynaptic in 60-65% of all synapses in which it was expressed pre- or postsynaptically, or both, in spinal laminae I-IV. Estimates for the gracile nucleus were higher (80%). No presynaptic NR1 was found in the ventroposterior thalamus. Because of the relative sparsity of presynaptic NR1 in terminals in laminae I and IIo and in terminals of peptidergic unmyelinated afferents, it is suggested that presynaptic NMDA receptors play a more significant role in modulation of mechanosensitive, innocuous input than in nociception.