The validation of a diffusion chamber comprising a donor and a receptor side separated by a cartilage membrane was undertaken according to the basic principles described by Peng et al. (1998). The study had three targets: first to evaluate the chamber as in vitro system by the examination of the diffusibility of compound through bovine cartilage samples; second the analysis of the affinity of compound (RS-130830) to cartilage; third to test the influence of two pre-incubation periods (one or three nights) of the cartilage samples. The validation of the chamber as in vitro system for the analysis of compound diffusibility and affinity to cartilage was performed using membrane slices of fresh bovine cartilage and a hydroxamic acid derivative (RS-130830) known as matrix metalloproteinase inhibitor (MMPI). The influence of the pre-incubation of cartilage was also examined. Compound concentrations in donor, receptor and membrane were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). Diffusion could be demonstrated after 6 h and finally 24 h incubation: the compound concentration in the receptor increased from 0 to 35 microM (mean) while it decreased in the donor from 200 to 144 microM (mean). We also found compound in the cartilage membrane (approximately 1.2 nmol (mean)). Pre-incubation of cartilage samples in culture buffer is suitable as a storage procedure, since the results on the donor side only were influenced significantly but not for the receptor and the cartilage affinity. Thus, the system could clearly reflect relevant properties of the tested compound with regard to its diffusibility and affinity to cartilage tissue.