Hyaluronan accumulates in ascites during intraperitoneal proliferation of TA3/St murine mammary carcinoma cells and at sites of their invasion of the peritoneal wall. To determine whether hyaluronan is functionally involved in these events, ascites tumor formation was compared in mice injected intraperitoneally with stable transfectants of TA3/St cells that overexpress soluble CD44, a hyaluronan-binding protein, versus in mice injected with transfectants expressing mutated soluble CD44 that does not bind hyaluronan. The soluble CD44 transfectants temporarily grew at a reduced rate within the peritoneal cavity, then went into G₁ arrest and were subsequently cleared from the peritoneum. However, transfectants overexpressing mutant soluble CD44 that does not bind hyaluronan exhibited similar ascites accumulation, growth rates, and cell-cycle profiles in vivo to wild-type and vector-transfected TA3/St cells, all of which continued to grow until the tumors became fatal. The soluble CD44-transfected TA3/St cells also failed to attach to and form tumors in the peritoneal wall. When grown in vitro in soft agar, the soluble CD44 transfectants exhibited a dramatic reduction in colony formation compared to wild-type, vector-transfected, and mutant soluble CD44-transfected TA3/St cells. Thus, perturbation of hyaluronan interactions by soluble CD44 has a direct effect on the growth characteristics of these tumor cells, leading to inhibition of anchorage-independent growth in vitro and ascites growth in vivo.