DNA from phytohaemagglutinin-stimulated lymphocytes which had been pulse-labelled for 1 min with [3H]deoxycytidine eluted as partially single-stranded DNA from columns of benzoylated napthoylated DEAE-cellulose. The label was transferred progressively into the double-stranded DNA fraction upon incubation in the presence of unlabelled deoxycytidine. The rate of transfer was slower in untreated lymphocytes from patients with megaloblastic anaemia than in corresponding control cells. A similar delay was also observed in normal lymphocytes treated with methotrexate or hydroxyurea. A close temporal correlation between the joining of Okazaki pieces (measured by alkaline sucrose gradients) and the transfer of the pulse label to double-stranded DNA suggested that the latter process represented the filling of gaps between Okazaki pieces. We suggest that this gap-filling step is retarded in megaloblastic anaemia and in cells treated with methotrexate or hydroxyurea.