Granulosa cells were isolated from follicular aspirates collected at ovum recovery for in vitro fertilization. Cells were cultured in a defined medium on artificial extracellular matrix (Matrigel) in the presence or absence of hCG as a model for corpus luteum function. Release of cells from this culture system is reduced by hCG and this effect may be mediated through an inhibition of extracellular matrix degradation. Using zymography and western blot analysis, we confirm the identity of matrix metalloproteinases-2 and -9 in culture media. Matrix metalloproteinase-9 was the predominant gelatinase in freshly prepared granulosa cells and in culture media, and also represented a major metalloproteinase component in homogenates of early and mid-luteal phase samples of corpora lutea. Quantitative analysis of matrix metalloproteinases in culture media, obtained throughout the 14 day culture period and expressed per μg of DNA, showed that matrix metalloproteinase-2, undetectable on day 2, rose throughout the culture period and that this rise was significantly inhibited by hCG. In contrast, matrix metalloproteinase-9 was clearly detectable on day 2 and remained relatively constant throughout much of the culture (day 2 to day 12) in the presence of gonadotrophin. Significantly increased production of matrix metalloproteinase-9 (day 6 to day 12) was evident in the absence of hCG. Our results provide further evidence for the hypothesis that the rescue of the corpus luteum in early pregnancy involves the maintenance of cellular function through the stabilization of the extracellular matrix.