Abstract Background Mucopolysaccharidosis I (MPS I) is a genetic disorder caused by deficiency of L-iduronidase (IDUA) activity. Heterozygote screening is a highly requested service by risk families; however, determination of IDUA activity alone is not sufficient to discriminate between heterozygotes and normal individuals because a significant overlap occurs between them. The aim of this study was to characterize the enzyme eluted from heterozygote's dried blood samples and determine if there are differences with that of normal individuals. Methods We determined Km, Vmax and the thermal stability of the enzyme at 50°C. Results Vmax from heterozygotes (7.28±2.72μmol/lblood/h) was significantly different than the obtained in controls (10.52±2.05μmol/lblood/h), while their Km were similar: 0.633±0.339mmol/l and 0.672±0.246mmol/l, respectively. After a 12 h pre-incubation period, IDUA activity in controls was significantly lower compared to heterozygotes. Conclusions IDUA eluted from dried blood spots of heterozygotes differs from that of controls in terms of Vmax and thermal stability. These parameters can be used as an important tool for the detection of carriers for MPS I. This is the first report describing a differential behavior of these parameters for a lysosomal enzyme obtained from dried blood.