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.