A method for measuring the expression of integrin subunits on the cell surface of knee ligament fibroblasts was developed with use of flow cytometry and immunofluorescence. The ligament cells exhibited uniform size and density, as shown by forward and side-scatter properties, and showed minimal nonspecific binding of isotype control antibodies compared with unstained cells. All cells expressed the α5 integrin subunit; lateral collateral ligament cells stained with antibody to α5 showed a mean fluorescence intensity 2-fold higher than that of medial collateral ligament cells, 1.5- fold higher than that of posterior cruciate ligament cells, and 3-fold higher than that of anterior cruciate ligament cells, indicating a greater expression of the α5 subunit by lateral collateral ligament cells than by medial collateral, posterior cruciate, and anterior cruciate ligament cells. All cells expressed the β1 integrin subunit; the expression by posterior cruciate ligament cells was 3-fold higher than that by medial collateral ligament or lateral collateral ligament cells and 5-fold higher than that by anterior cruciate ligament cells. All cells expressed the β3 integrin subunit; the expression by posterior cruciate ligament cells was 1.5, 3, and 4.5-fold greater than that by lateral collateral anterior cruciate, and medial collateral ligament cells, respectively. Our data suggest there is a differential expression of integrin subunits in knee ligament fibroblasts, and this in part may explain differences in their attachment and adherence to extracellular matrix molecules.