Thyroid hormone plays an important role in bone development and metabolism. We used a polymerase chain reaction (PCR)-based mRNA differential display (DD) analysis to obtain a profile of thyroid hormone-responsive genes in osteoblast-like cells (ROS 17/2.8). ROS 17/2.8 cells were treated with 10(-8) M triiodothyronine (T(3)) for 2 and 24 hours. Total RNA was isolated, reverse-transcribed, and amplified using a total of 72 combinations (2 hours) and 240 combinations (24 hours) of 5' and 3' primers. At the 2-hour time point, 1 true-positive novel clone was identified and shown to be the mitochondrial gene, subunit 6 of ATP synthase (ATPase-6). At the 24-hour time point, 3 differentially expressed (DE) mRNAs were confirmed as true-positives including; nonmuscle alkali myosin light chain (NM aMLC), ATPase-6, and one novel clone. T(3)-induction of ATPase-6 mRNA in ROS 17/2.8 cells was seen at 2 and 4 hours, but was maximal at 24 hours (2.1-fold). T(3) induction of ATPase-6 mRNA was increased to fourfold in ROS 17/2.8 cells cultured at a low density. NM aMLC mRNA was modestly upregulated by T(3) in ROS 17/2.8 cells by 1.4-fold, and induction was augmented at low cell density to 1.7-fold. T(3) action on NM aMLC and on the mitochondrial gene ATPase 6, represent novel targets and potential mediators of thyroid hormone action on bone. Cell type, and the extent of cell differentiation, influences T(3) regulation of genes in osteoblast-derived cells.