The double fluorescent retrograde labeling method was used to examine the distribution of spinothalamic tract (STT) cells that project to the medial and lateral thalamus in the cat. Injections of one fluorescent tracer (Fast Blue or Diamidino Yellow) were made throughout the lateral thalamus and injections of the other tracer were made in the medial thalamus at sites extrapolated from recording track coordinates. Survival times were successively extended (up to 5 weeks) in order to maximize labeling in both the cervical and lumbosacral spinal cord. On average, over 2,000 labeled contralateral STT cells were counted in serial sections from segments C5-7 and L5-S2. Numerical variability of the order of a factor of two was attributable to inherent differences between individual animals. The total number of cells labeled with fluorescent tracers was comparable to the number labeled with horseradish peroxidase in control cases, although there were significant differences between the laminar distributions of labeling produced by the two methods. Injections made anterior to the thalamus to control for labeling due to leakage or passing fibers did not produce substantial spinal labeling. The laminar distribution of fluorescent dye-labeled STT cells was consistent; about half (47%) were located in lamina I, 8% were in lamina V, 5% in lamina VI, 20% in lamina VII, and 20% in lamina VIII. The proportions of STT cells in laminae I and V were higher in cervical segments (57% and 12%, respectively) than in lumbosacral segments (38% and 6%). The dominant contribution of lamina I cells to the STT thus revealed by the fluorescent tracers is striking. The proportions of STT cells labeled from the medial and the lateral thalamus varied with segmental and laminar location and with injection placement. The majority (62%) of STT cells in most cases projected only to the medial thalamus, 25% projected only to the lateral thalamus, and 13% projected to both. The STT cell populations in laminae I, VII, and VIII each displayed this common projection pattern. In contrast, cells in laminae V and VI projected predominantly to the lateral thalamus. Twice as many STT cells in lamina I (19%) projected to both the medial and the lateral thalamus as from other laminae. A greater proportion of laminae V-VIII STT cells in segments L5-6 projected to the lateral thalamus, and in S1-2, more projected to the medial thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)