Space-flown rats show a number of flight-induced changes in the structure and function of pituitary growth hormone (GH) cells after in vitro postflight testing (W. C. Hymen, R. E. Grindeland, I. Krasnov, I, Victorov, K. Motter, P. Mukherjee, K. Shellenberger, and M. Vasques. J. Appl. Physiol. 73, Suppl.: 151S-157S, 1992). To evaluate the possible effects of microgravity on growth hormone (GH) cells themselves, freshly dispersed rat anterior pituitary gland cells were seeded into vials containing serum +/- microM hydrocortisone (HC) before flight. Five different cell preparations were used: the entire mixed-cell population of various hormone-producing cell types, cells of density < 1.071 g/cm3 (band 1), cells of density > 1.071 g/cm3 (band 2), and cells prepared from either the dorsal or ventral part of the gland. Relative to ground control samples, bioactive GH released from dense cells during flight was reduced in HC-free medium but was increased in HC-containing medium. Band 1 and mixed cells usually showed opposite HC-dependent responses. Release of bioactive GH from ventral flight cells was lower; postflight responses to GH-releasing hormone challenge were reduced, and the cytoplasmic area occupied by GH in the dense cells was greater. Collectively, the data show that the chemistry and cellular makeup of the culture system modifies the response of GH cells to microgravity. As such, these cells offer a system to identify gravisensing mechanisms in secretory cells in future microgravity research.