As an extension of the previously reported observation concerning the existence of NAD-dependent 5,10-methylenetrahydrofolate dehydrogenase in transformed cells a variety of tissues and cell lines have been assayed for this activity. This activity was found in all assayed transformed cells. Results with rat liver derived epithelial (RLE) cells transformed with a series of oncogenes (v-raf, v-raf/v-myc (J2), v-myc (J5), and v-Ha-ras (pRNR16)) indicated that expression of activity correlates with the extent of transformation and was independent of the oncogene used for transformation. Compared to previously reported values for normal tissue, surprisingly high levels of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase were found in the rat adrenal cortex. This activity was not seen in mouse or bovine adrenal. Enzymatic activity was also detected in mouse bone marrow and was strain dependent. The levels of activity in mouse bone marrow were lower than previously reported. The NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase activity in rat adrenal and RLE cells may represent tools for studying the regulation of expression of this activity.