Estrogens have been shown to be essential for maintaining a sufficiently high bone mineral density and ERα expression has been demonstrated in bone cells. Recently, a novel estrogen receptor, estrogen receptor β (ERβ) has been identified. Here we demonstrate that also ERβ is expressed in human osteoblasts, and that ERα and ERβ are differentially expressed during human osteoblast differentiation. ERβ mRNA expression increased gradually during osteoblast culture, resulting in an average increase of 9.9 ± 5.3 fold (mean ± S.D., n = 3) at day 21 (mineralization phase) as compared to day 6 (proliferation phase). In contrast, ERα mRNA expression levels increased only slightly until day 10 (2.3 ± 1.7 fold) and then remained constant. The observed differential regulation of ERα and β is suggestive for an additional functional role of ERβ to ERα in bone metabolism.