In the rat lumbar spinal cord the major supraspinal targets for lamina I projection neurons are the caudal ventrolateral medulla (CVLM), lateral parabrachial area (LPb) and periaqueductal grey matter (PAG). In this study we have estimated the number of lamina I neurons retrogradely labelled from each of these sites in the L4 segment, as well as the proportion that can be labelled by injecting different tracers into two separate sites. Our results suggest that this segment contains approximately 400 lamina I projection neurons on each side, and that approximately 85% of these can be labelled from either the CVLM or the LPb on the contralateral side. Around 120 lamina I cells in L4 project to the PAG, and over 90% of these cells can also be labelled from the CVLM or LPb. Most lamina I neurons projecting to CVLM or LPb are located in the contralateral dorsal horn, but in each case some cells were found to have bilateral projections. We also examined horizontal sections to investigate morphology and the expression of the neurokinin 1 (NK1) receptor in cells labelled from CVLM, LPb or PAG. There were no consistent morphological differences between these groups, however, while cells with strong or moderate NK1 receptor-immunostaining were labelled from LPb or CVLM, they seldom projected to the PAG. These results suggest that many lamina I cells project to more than one site in the brain and that those projecting to PAG may represent a distinct subclass of lamina I projection neuron.