Hydra interstitial stem cells continuously give rise to daughter stem cells as well as precursors for nerve and nematocyte differentiation. Growth of the stem cell population is controlled by the self-renewal probability (Ps): Ps is the fraction of stem cell daughters that remain stem cells in each generation. We have determined Ps for Hydra interstitial stem cells by using a novel technique based on the cell conposition of clones. Stem cell clones were grown in aggregates of nitrogen mustard-inactivated Hydra tissue. They contain several hundred cells after 14 days of growth, including stem cells, differentiating nematocytes, and differentiating nerve cells. Clone size, size variability, and the ratio of differentiating cells to stem cells are sensitive measures of Ps. We have prepared standard curves relating these parameters to Ps, using computer simulations of clone growth. Comparisoon of the experimentally observed parameter of clones to these curves indicates that Ps decreases from 0.8 in 5- to 6-day clones to 0.6 in 10- to 12-day clones. The decrease in Ps coincides with the increase in clone size and suggest that Ps may be regulated by the density of stem cells in clones. Such a mechanism could be responsible for the observed homeostasis of stem cell populations in vivo.