Liver and blood δ-aminolevulinic acid dehydratase (ALA-D) inhibition by exposure to sub-lethal lead concentrations over time in Nile tilapia (Oreochromis niloticus) were investigated. All three lead concentrations (1 mg kg−1, 10 mg kg−1 and 100 mg kg−1) significantly inhibited ALA-D activity in blood (319±29.2; 180±14.6 and 172±19 µmols−1 h−1 L−1 respectively) and liver (302±5.84; 201±41.4 and 93±22.1 µmols−1 h−1 L−1) 24 h after injection relative to controls (blood: 597±37.0 µmols−1 h−1 L−1; liver: 376±23.1 µmols−1 h−1 L−1). Blood ALA-D was greatly inhibited in all but the highest lead dose. Fish were then exposed to 1 mg kg−1 lead for 9 days, and presented short-term hyperglycemia, decreased hemoglobin and hematocrit values and time-dependent blood ALA-D activity inhibition, corroborating blood ALA-D activity as being more suitable for investigating lead effects, showing dose and time-dependent ALA-D inhibition after lead exposure. The results of the present study also demonstrated that fish size affects blood ALA-D activity, as fish from the 24-h assay, which were slightly smaller (approximately 200 g), showed higher ALA-D inhibition in response to lead exposure when compared to the fish from the 9-day assay (approximately 500 g). Thus, fish size should always be taken into account both in the field and in laboratory settings, and efforts should be made to obtain uniform fish size samples for biomarker studies.