We report the distribution of choline acetyltransferase (ChAT) activity in the laminae of the rat olfactory tubercle. Within its posterior medical portion, the tubercle contains three parallel histological laminae that can be separated by cutting tangential sections from frozen tissue. ChAT was measured in homogenates of consecutive sections (16 micrometers) cut parallel to these laminae. The distribution of ChAT activity, as a function of tubercle depth, showed a broad peak centered at 500 micrometers from the ventral surface of the brain. Enzyme activity measured at this depth (85 pmol acetylcholine formed/microgram protein/h) was 2 1/2 times greater than that measured in the outermost, plexiform, layer. Stereotaxic injections of kainic acid (1 microgram in 1 microliter) made directly into the tubercle were used to eliminate intrinsic neurons. Three days after injection, histological examination revealed the almost total absence of neuronal cell bodies and the proliferation of glial cells. The greatest decreases in ChAT activity (50%) were seen at depths of 300-600 micrometers whereas no loss of activity occurred in the plexiform layer.