Chromatography on benzoylated naphthoylated DEAE-cellulose has been used to fractionate fully double-stranded from partially single-stranded DNA molecules. DNA was extracted from phytohemagglutinin-stimulated lymphocytes from patients with megaloblastic anemia resulting from vitamin B12 or folate deficiency after pulse-labeling the cells with [3H]thymidine for 5 min and chasing in unlabeled medium for 24 h. No gross accumulation of partially single-stranded material was observed in the DNA of these cells when compared with DNA from similarly labeled control cells obtained by the addition of 5-formyl tetrahydrofolic acid to the culture medium. When DNA from lymphocytes labeled with a 5-min pulse of [3H]thymidine and sheared to fragments of an average length of 18 micrometer was chromatographed on benzoylated naphthoylated DEAE-cellulose, approximately 80% of the label was recovered in the partially single-stranded fraction. After chasing in unlabeled medium the label was progressively transferred to the double-stranded fraction over a period of 2--3 h. The rate of transfer was slower in megaloblastic lymphocytes than in controls. The difference in rate suggested a slower rate of replication fork movement in megaloblastic lymphocytes and so the density shift technique of Painter and schaeffer (J. Mol. Biol. 45: 467--479, 1969) was used to measure the fork rate directly. [3H]Deoxycytidine was used as the labeled nucleoside to avoid possible complications arising from [3H]thymidine labeling of megaloblastic cells. Investigations on the lymphocytes from four patients showed that the replication fork rate in vitamin-treated control lyphocytes was about 1 micrometer/min. The fork rates in the corresponding untreated cells were invariably lower and rates ranging from 40 to 92% of those of controls were observed. Normal lymphocytes treated with the deoxynucleotide pool-depleting drugs methotrexate or hydroxyurea displayed defects in DNA synthesis similar to those of untreated megaloblastic lymphocytes. We propose that the delayed DNA replication fork movement in cells of patients with megaloblastic anemia results from impaired biosynthesis of DNA precursors.