Recent studies have revealed that osteocytes play multiple important physiological roles. To analyze osteocyte functions in detail, an in vitro experimental system for primary osteocytes would be useful. Unfortunately, osteocytes tend to dedifferentiate and acquire osteoblast-like features even when the cells are cultured in three-dimensional (3D) collagen gel. Therefore, it is desirable to establish osteocyte culture conditions that prevent dedifferentiation over longer periods. In this study, we obtained systematic information about the influence of culture conditions on osteocyte differentiation states. Fetal bovine serum (FBS) concentrations from 0.1 to 0.5 % in 3D culture matrix did not significantly influence the expression of osteocyte markers. On the other hand, addition of Matrigel to the culture matrix significantly enhanced the expression of Rankl and late osteocyte markers such as Sost and Fgf23. Matrigel addition also inhibited upregulation of Opg and early osteocyte markers such as Dmp1 and Gp38. These effects on osteocyte properties were maximal at a Matrigel culture matrix content of 50 %. Matrigel addition to the matrix also increased dendritic process extension by osteocytes. In addition, Matrigel addition significantly stimulated tartrate-resistant acid phosphatase activity in co-culture with bone marrow macrophages. Among the conditions tested, 50 % Matrigel and 0.2 % FBS in type I collagen matrix were optimal for culture of primary osteocytes.