Developmental aspects of the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4-PteGlu) were studied in suckling (14-day-old), weanling (22-day-old), and adult (90-day-old) rats by use of the intestinal everted-sac technique. Mucosal-to-serosal transport of 0.5 microM 5-CH3H4PteGlu was linear with time for 40-min incubation and occurred at a rate of 0.035, 0.032, and 0.010 nmol X g initial tissue wet wt-1 X min-1 for suckling, weanling, and adult rats, respectively. The transport of 5-CH3H4PteGlu in all age groups was pH dependent (maximal at pH 6) and was higher in the jejunum than in the ileum. In all age groups the transport of 5-CH3H4PteGlu occurred by an active carrier-mediated system. The system was saturable; energy, temperature, and Na dependent; inhibited by structural analogues; and capable of accumulating the substrate against a concentration gradient. Kinetic parameters of the transport process, however, showed some difference. A progressive decrease in Vmax was observed from suckling to weanling to adult rats (5.1, 3.7, and 0.8 nmol X g initial tissue wet wt-1 X 30 min-1, respectively), while apparent Kt was similar (2.2, 1.73, and 1.79 microM, respectively). This study demonstrates that the transport system of 5-CH3H4PteGlu in the rat is fully developed at the suckling age. The results also suggest that the activity and/or the number, but not the affinity, of the transport carriers decrease with maturation.