The optimization of the method for acid alpha-glucosidase determination (EC 3.2.1.3) in human urines, employing the synthetic substrate 4-nitrophenyl-alpha-D-glucopyranoside, is reported. Storage conditions of the specimens and their pretreatment were particularly investigated. The precision of the whole analytical procedure (including gel filtration) is good (within-run CV = 7.4% for normal samples and 3.7% for elevated ones). The correlation with the method using maltose as substrate is excellent (y = -0.01 + 0.13 x; r = 0.9893).