The uptake and turnover of 3H-uridine by periosteal and endosteal osteoblasts were assessed autoradiographically. Five-week old mice were injected with 5 m̈CI of 3H-uridine (Sp. Act. 21 Ci/mM) per gram of body weight and killed at intervals varying from 15 minutes to one month post-injection. Femora were processed histologically and autoradiographs were prepared from 5 m̈m decalcified sections exposed for 36 days. Autoradiographic grains were counted over the nucleus and the cytoplasm of 600 periosteal and 600 endosteal osteoblasts per time period. 3H-Uridine was initially rapidly incorporated into the nucleus attaining peak activity one hour after injection. Incorporation of label into cytoplasmic RNA was maximum at one to two days. At one month grain counts were at minimum values. The loss was more gradual from the nucleus than from the cytoplasm. At earlier time periods the nucleus showed a higher label than the cytoplasm. At 8 to 16 hours the label was evenly distributed and at later time periods the cytoplasm exhibited a higher grain count. Dia-physeal periosteal osteoblasts and metaphyseal endosteal osteoblasts differed in both the quantity of label and the rate of utilization. Endosteal cells exhibited almost twice the activity. RNA biosynthesis appears to be significantly higher in endosteal cells than periosteal cells thus reflecting differences in protein synthesis by these two cell compartments.