Mice of the BALB/cJ strain have more neurons and greater tyrosine hydroxylase (TH) activity in the midbrain than mice of the CBA/J strain (Baker, H., T. H. Joh, and D. J. Reis (1980) Proc. Natl. Acad. Sci. U.S.A. 77: 4369–4373). To determine whether the strain differences in dopamine (DA) neuron number and regional TH activity are more generalized, regional TH activity was measured and counts of neurons containing the enzyme were made in the hypothalamus of male mice of the BALB/cJ and CBA/J strains. TH activity was measured in dissections of whole hypothalamus (excluding the preoptic area), the preoptic area containing a rostral extension of the A14 group, the mediobasal hypothalamus containing the A12 group, and the mediodorsal hypothalamus containing neurons of the A13 and A14 groups. Serial sections were taken and the number of DA neurons was established by counting at 50- to 60-microns intervals all cells stained for TH through each area. In conjunction with data obtained biochemically, the average amount of TH per neuron was determined. In all areas, TH activity in CBA/J mice was significantly less (p less than 0.001) than in BALB/cJ mice, ranging from 48% in the mediobasal hypothalamus to 71% in the medial and dorsal hypothalamus. The number of TH-containing neurons was also significantly less in the CBA/J strain (p less than 0.001), ranging from 49% in the preoptic area to 74% in the mediobasal hypothalamus (MBH). With the exception of the MBH, enzyme activity per neuron was similar in both strains. In the MBH, strain differences in TH activity were greater than those for neuron number, resulting in less TH activity per neuron in the CBA/J strain. The results suggest that strain differences in the number of DA neurons are widespread and involve DA systems throughout the brain. Therefore, differences in whole brain TH activity cannot be attributed only to differences in specific regions. Our findings further support the view that the number of neurons of a specific chemical class may be under genetic control.