In a previous study, we presented evidence that the synthesis of hyaluronan by hypertrophic chondrocytes is one of the principal factors driving the interstitial expansion of the growth plate (Pavasant et al., J. Cell Sci. 109: 327-334, 1996). To test this possibility further, we used two different approaches to examine the effects of hormones on the production of hyaluronan in the growth plate. In the first approach, we examined the growth plate of the lit/lit mouse that lacks growth hormone and found that its hypertrophic lacunae were smaller and contained less hyaluronan than those of wild type mice. Moreover, the ratios of hyaluronan staining density to total area of the lacunae were similar for the lit/lit and the wt/wt mice, indicating that the amount of hyaluronan is directly related to lacuna size. In the second approach, we examined the effects of hormones on segments of the epiphysial growth plate placed in organ culture. Under normal culture conditions, a band of hyaluronan staining progressed across the length of the growth plate, reflecting the maturation of chondrocytes into the hypertrophic stage. When insulin-like growth factor-1, a factor known to promote chondrocyte maturation, was added to the culture medium, the production of hyaluronan and the enlargement of the lacunae were stimulated. In contrast, when either parathyroid hormone or transforming growth factor-beta 1, both of which inhibit chondrocyte differentiation, was added to the medium of cultured segments, new pericellular hyaluronan was not detected and the lacunae did not enlarge. Taken together, these results indicate that factors that either stimulate or inhibit the maturation of epiphysial chondrocytes have a corresponding effect on the production of hyaluronan. This, in turn, further supports the importance of hyaluronan in the process of lacuna enlargement.