To gain insight into the mechanisms responsible for differentiation of hippocampal neurons growing in vitro, the effects of estrogen on neuritic development and on activity and distribution of isoforms of the Na, K-ATPase, were evaluated. Dissociated cells from hippocampi of 19-day-old rat fetuses were raised for 5 days in the presence or absence of 100 nM estradiol-17 beta (E2) in minimum essential medium supplemented either with 10% untreated fetal calf serum (MEM-10) or with 10% fetal calf serum previously adsorbed with dextran-activated charcoal (MEM-10-Cha). Cultures in MEM-10 showed larger neuritic length and increased levels of Na, K-ATPase activity than cultures in MEM-10-Cha. In cells cultured in MEM-10 medium, the addition of E2 resulted in selective enhancement of axonal length with a concomitant increase in the alpha-2 isoform of the Na, K-ATPase, whereas a decrease was found in the form most sensitive to ouabain; the total enzymatic activity remained unchanged. Conversely, in cultures raised in MEM-10-Cha, E2 did not affect Na, K-ATPase activity or neuritogenesis. These results show that two presumably independent probes of cellular differentiation of hippocampal neurons (i.e., neuritogenesis and patterns of Na, K-ATPase activity) were concurrently regulated by E2 and that such regulation depended on interaction with factor(s) present in calf serum. The well-known neuritogenic effect of E2 is hereby extended to hippocampal neurons, although for these cells it seems to be restricted to axons.