It has been hypothesized that the corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) become hyperactive with age, and even more so in Alzheimer's disease. This hyperactivity could be due to an increased production of CRH per neuron, or an increased number of PVN neurons producing CRH, or both. As a first step in elucidating which of these biological mechanisms might be operative, we have estimated the absolute number of CRH immunoreactive neurons in the PVN of 10 human control subjects between 36 and 91 years of age and 10 Alzheimer patients between 40 and 97 years of age. CRH neurons were immunocytochemically detected in 6 microns paraffin sections with the aid of a highly specific monoclonal antibody to CRH. The antibody signal was amplified by the biotin-streptavidin and alkaline phosphatase methods. The absolute number of CRH neurons in the PVN was obtained by multiplying the number of CRH neurons in a unit volume (NV) by the total volume of the PVN. Two different methods were used to estimate the NV: an unfolding method and a disector method (about three times more time-consuming). Compared to the disector, the unfolding method consistently yielded a lower cell number for all patients by 38% (+/- 2.8%; mean +/- SEM). However, both methods yielded an increase in the absolute number of CRH neurons in control and Alzheimer patients with age. No statistically significant difference in the absolute number of CRH neurons was found between control and Alzheimer patients with both methods. The age-dependent increase in the absolute number of CRH neurons within the PVN of both control and Alzheimer patients is interpreted as a sign of activation of the CRH neurons with age.