Several functional subclones of rat anterior pituitary cells were established from our 2A8 clone which apparently contains a heterogenous population of committed and uncommitted cells. On the basis of the hormones secreted into the culture media, as measured by radioimmunoassay, these subclones were divided into four categories, i.e., subclones which secrete (1) ACTH only, (2) prolactin only, (3) prolactin and GH or (4) ACTH, prolactin and GH. None of the subclones produced detectable amounts of thyrotrophic or gonadotrophic hormones. Subclones which secrete a single hormone have shown no change in the type of hormone produced, indicating that these subclones were each derived from a committed cell. The cells of all subclones exhibit a normal diploid karyotype and show good growth characteristics. The cells of the different subclones can be classified by phase contrast microscopy into four categories. However, no clear-cut ultrastructural features have been observed which can be correlated with the different categories of subclones. On the basis of the results a hypothesis is proposed relative to the functional cytodifferentiation of anterior pituitary cells.