In preterm neonates the immune system is thought to be less developed at birth, but very little is known about the actual size of lymphocyte subpopulations, and even less about the maturation of these subpopulations during the first months after a premature birth. To evaluate the development of lymphocyte subpopulations in preterm infants during the first 3 months after birth, we performed a prospective longitudinal study in two hospitals in the Netherlands. Preterm neonates (n = 38) of all post-menstrual ages were included and blood samples were taken from cord blood, and at 1 week, 6 weeks, and 3 months. Lymphocyte subpopulations were measured by four-colour flow cytometry. The data were compared with follow-up data obtained in healthy term neonates (n = 8), and with single samples from school age children (n = 5) and adults (n = 5). Overall, we found a similar pattern of post-natal development of lymphocyte subpopulations in the term and preterm infants. Both B lymphocytes and helper and cytotoxic T lymphocytes mainly consist of naive cells at birth and during the 3 months of follow-up in all neonatal age groups. So, the preterm immune system seems to be able to generate an outburst of naive T and B lymphocytes from the thymus and bone marrow within the same time span after the start of post-natal antigenic stimulation from the environment as the term immune system, but, with lower post-menstrual age, the absolute counts of naive helper T lymphocytes are lower.