We previously reported that versican, a large chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding was strongly inhibited by the chondroitinase digestion of versican or by the addition of soluble chondroitin sulfate (CS) B, CS E, or heparan sulfate. Furthermore, these glycosaminoglycans (GAGs) could bind directly to the chemokines that bind versican. Thus, versican appears to interact with chemokines via its GAGs. We next examined if versican or GAGs affect secondary lymphoid tissue chemokine (SLC)-induced integrin activation and Ca(2+) mobilization in lymphoid cells expressing a receptor for SLC, CC chemokine receptor 7. Interestingly, whereas heparan sulfate supported both alpha(4)beta(7) integrin-dependent binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-IgG and Ca(2+) mobilization induced by SLC, versican or CS B inhibited these cellular responses, and the extent of inhibition was dependent on the dose of versican or CS B added. These findings suggest that different proteoglycans have different functions in the regulation of chemokine activities and that versican may negatively regulate the function of SLC via its GAG chains.