Several studies have shown that a certain subpopulation of middle-aged rats have an attenuated preovulatory LH surge, and that this is an early indicator of a general decline in reproductive cyclicity. The pituitary glands of middle-aged animals with attenuated LH surges have been shown to be significantly less responsive to GnRH than those from young (Y) animals. The present study was designed to investigate the relationship between the in vivo LH surge and the in vitro secretory capacity of dispersed anterior pituitary gland cells from the same animals. Individual LH-secreting cells were also studied with respect to their LH secretory capacity and proportionate numbers in the pituitary gland using reverse hemolytic plaque assay (RHPA). Two groups of animals, young (4–5 months old) and middle-aged (10–12 months old), were sampled on the afternoon of proestrus to determine the amplitudes of their preovulatory LH surges. The middle-aged animals were then classified as either normal (MAN) or attenuated (MAA) with regard to the peak levels of the surges. On the next proestrus, dispersed anterior pituitary gland cells were prepared for either perifusion or RHPA. Perifused anterior pituitary gland cells were exposed to three 2.5-min pulses of GnRH (30 nM) at 60-min intervals. In the RHPA, red blood cells were treated with protein-A to allow coating with rabbit antiovine LH-beta, and then co-incubated with anterior pituitary gland cells. The cells were then treated with one of several concentrations of GnRH (0, 0.1, 1.0, 10, or 100 nM). GnRH responsiveness of dispersed anterior pituitary gland cells from MAA animals was decreased compared to cells from Y animals. Comparison of MAA to Y animals showed that proportional LH release was greater in vitro than in vivo. This corroborated results from previous studies using perifused pituitary fragments. Responsiveness of individual LH-secreting cells, as indicated by mean plaque area, was not different between Y and middle-aged rats at 12.00 h. However, the percentage of anterior pituitary gland cells from MAA animals that formed LH plaques were significantly lower than those from Y animals at 12.00 h, under both basal and GnRH-stimulated conditions. These results verify that the pituitary glands of MAA animals are less responsive than those of Y animals, and imply potential changes in GnRH output from the hypothalamus in vivo. They also indicate that the decreased responsiveness of pituitary tissue from MAA animals may be due more to a decrease in the percentage of anterior pituitary gland cells that release LH than to a decrease in LH responsiveness of individual cells.