The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.