The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with [³⁵S]sulfate and [³H]proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of ³⁵S- and ³H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of ³⁵S- and ³H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of ³H- and ³⁵S-labeled macromolecules that was accompanied by an increase in the rate of loss of [³H]hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The ³⁵S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (~15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and ³⁵S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of ³⁵S-labeled macromolecules. This study provides a framework for elucidating the biophysical mechanisms involved in the redistribution, catabolism, and loss of macromolecules during cartilage compression.