Hyaluronidase activity was compared in embryonic chick cardiac cushion and noncushion segments, as well as in cultures of mesenchyme derived from cardiac cushion endocardium (cushion tissue-enriched cultures) and in cultures of myocardial cells at stages critical to heart valve and septum development. Enzyme levels were higher in both heart tissue regions at periods of active cushion tissue mesenchyme migration than after migration ceases, and higher in the cushion region than in the noncushion region at both periods. Hyaluronidase was measured in cells and medium in both types of cultures, with five times greater activity found in the myocardial cultures. The cardiac hyaluronidase from cells and medium of both culture types had an estimated molecular weight of 41,000 to 44,000 and degraded hyaluronate and, to a lesser degree, chondroitin sulfate, at an acidic pH optimum. Ion-exchange chromatography demonstrated that in both culture types, a proportion of the secreted enzyme was more acidic than that found in the cell layer. These studies indicate the potential for hyaluronate degradation by the major cell types present in the developing heart at early stages and that the enzyme responsible is probably a lysosomal enzyme. Therefore, hyaluronate internalization is a likely requirement for degradation, and thus, the turnover of hyaluronate in developing heart valves is more complex than the extracellular degradative process suggested by histochemical data.