Administration of ammonia caramel color (AC) to rats may decrease blood lymphocyte counts, specifically in rats fed a diet low in vitamin B6. This effect is associated with 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazole (THI). To characterize and compare the effects of AC and THI and to study the influence of dietary pyridoxine, two studies in rats were conducted. Weanling rats fed a diet containing 2-3 ppm pyridoxine and exposed to 4% AC or 5.72 ppm THI in drinking water for 4 weeks showed reduced cell numbers in spleen and popliteal lymph nodes, as well as in the blood. Flow cytometric analyses demonstrated a comparable reduction in B and T lymphocytes. In blood, spleen, and popliteal lymph nodes, CD4+ lymphocytes were more reduced than CD8+ cells. The number of bone marrow cells was not affected. Although thymus weight and cell number were not affected either, a decreased cortex over medulla area ratio and an increase in medullary cell density largely due to an increase in CD4+ thymocytes was observed. Decreased numbers of ED2+ macrophages were observed in the thymic cortex and in the spleen red pulp. All effects observed were either less pronounced or absent in a study with rats fed a diet containing 11-12 ppm pyridoxine. The effects of AC and THI on the immune system were similar. Whereas AC exposure was associated with changes in vitamin B6 status, THI did not induce obvious effects on vitamin B6 parameters. It is proposed that the effects of AC and THI on the immune system are not caused by a disturbance of vitamin B6 metabolism, but may in fact result from a disturbance of a specific PLP-dependent process.