The aim of this study was to evaluate the effect of nicotine on hepatic and blood d-ALA-D (d-aminolevulinic acid dehydratase) activity of rats treated with nicotine. Thirty-six male Wistar rats were housed in groups of six per cage, had free access to food and water, and were maintained on an inverted 10-14-h light-dark cycle. The animals were treated with saline 0.9 % or nicotine 5 mg/kg/day (s.c.) (5 consecutive doses at 8, 10, 12, 14 and 16:00 h) during 28 or 56 days. The rats were weighed, anesthetized and killed by decapitation about 1 h and 30 min after the last dose of saline or nicotine. For ex vivo d-ALA-D activity assays, the liver was quickly removed and placed on ice, and was homogenized in proportion 1:7 (w/v) in saline 150 mM. The homogenate was centrifuged at 8000 ´ g for 40 min at 4^oC. The supernatant fraction was used in the enzyme assays. The blood was colleted in heparinized tube and the hemolysis was carried out in proportion 1:4 (v/v) in water, with agitation by 10 min in ice bath. The material obtained was used for biochemical assay. d-ALA-D activity was assayed according to the method of Sassa (1982) with a molar absorption coefficient of 6.2 ´ 10⁴ for Ehrlich-porphobilinogen salt (555 nm). The liver and blood samples were incubated at 39^oC for 40 and 120 min, respectively. The specific activity was expressed as nmol of PBG formed per hour per mg protein. All samples were run in triplicate. The statistical analysis shows that the exposure to nicotine 5 mg/kg/day by 28 or 56 days did not inhibit the hepatic and blood d-ALA-D activity of rats. The results of liver and blood of both sources were similar. However, these results differ of those obtained in in vitro experiment, where the nicotine inhibited the d-ALA-D activity from these two sources. The absence of the effect ex vivo may be related at least to two possibilities: low levels of nicotine reached in vivo; or a possible inhibition induced by low concentrations of the alkaloid (in vivo) to be reverted by saturating concentration of substrate assayed ex vivo.