The β1 integrin subfamily, alternatively called very late activation antigen (VLA), has been implicated in various cellular functions. In this study, we generated a mAb against the mouse β1 subunit (CD29) to examine the functional property of mouse VLA proteins. After immunization with affinity-purified mouse VLA-4 (α4β1), a hamster mAb, HMβ1‐1, was established by screening mAb that reacted with α4-negatlve neuroblastoma C1300. The antigen defined by HMβ1‐1 was widely distributed in various mouse cell lines and HMβ1‐1 immunoprecipitated a 110‐120 kDa protein common to VLA-1 and VLA-6, indicating that HMβ1‐1 recognizes the β1 subunit of mouse integrins. We then examined the inhibitory effect of HMβ1‐1 on VLA-dependent cell adhesion and activation. HMβ1‐1 blocked the adhesion of mouse tumor cell lines to extracellular matrix proteins including collagen, laminin and fibronectin. Moreover, splenic T cell proliferation induced by anti-CD3 mAb and allogeneic mixed lymphocyte response were strongly inhibited by HMβ1‐1 in combination with an anti-LFA-1 mAb. We conclude that HMβ1‐1 reactive with mouse CD29 can inhibit VLA-dependent cellular functions and, thus, would be useful for studying the physiological role of β1 integrins in vivo.