Lymphoid tissue inducer (LTi) cells have a well established role in secondary lymphoid tissue development. Here, we report on the heterogeneity of LTi cells based on their CD4 and chemokine receptor expression. The CD4− LTi-cell population has a similar phenotype to the CD4+ population, with similar chemokine-receptor-expressing subsets. In both embryonic and adult spleen the CD4− LTi-cell population is comparable as a proportion of total splenocytes to its CD4+ counterpart. In contrast, different proportions of CD4+ and CD4− LTi cells are found in different lymph nodes. Both CD4+ and CD4− LTi cells share the anatomical location and are associated with vascular cell adhesion molecule-1-positive stromal cells in spleen and lymph nodes. The numbers of both CD4+ and CD4− LTi cells in adult spleen are augmented in the presence of B cells. With the exception of CD4, there is a strong correlation coefficient (0·89) for gene expression between the two populations. Polymerase chain reaction analysis of individual CD4+ and CD4− LTi cells shows that a similar proportion in embryonic and adult spleen co-expressed both CXCR5 and CCR7 or CXCR5 alone: 84·6% for adult CD4+ and 87·6% for adult CD4−; 95·3% for embryonic CD4+ and 91·5% for embryonic CD4−. Consistently fewer CCR7 single-positive cells were found in the CD4+ and CD4− fractions in the embryo.