Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the α1β1 and α2β1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and β-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat α1 and/or α2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2–treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-Collagen-I or both anti–integrin-α1 and -α2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5–1.8% control) of these cells. In all cases, adding anti-α1 or anti-α2 antibodies separately produced partial effects, while their combined effect approached that of anti-Collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by α1 and α2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.