Opinions differ concerning when theca cells arise during follicular growth. Theca cells cannot be distinguished from ovarian stroma cells on the basis of morphology alone. However, long-term continuous infusion of [3]thymidine (3H-TdR) thoroughly labels theca cells of large and medium-sized follicles whereas most stromal cells remain unlabeled. The objective of this study was to determine when, during the course of follicular development, labeled, squamous cells located just outside the follicular basement membrane (putative theca cells) first become apparent. Rats were given infusions of 3H-TdR for various periods of time. Autoradiographs were prepared from 2-microns-thick plastic sections of ovaries. Morphologically recognizable theca cells of large and medium-sized follicles were sharply delineated from surrounding stroma cells by their labeling pattern. Although unilaminar follicles lacked morphologically distinct theca layers, they all were closely enveloped by labeled cells just outside the basement membrane. Follicles with a few as 4-5 granulosa cells in cross section often had labeled cells on the convex surface of their basement membranes. These observations suggest the possibility that theca cells may already be present at the very outset of follicular growth.