Lymphoblasts from 61 untreated patients with acute lymphoblastic leukemia (ALL), classified according to the French-American-British (FAB) morphologic classification, were studied for cytochemical characteristics and membrane surface markers. Seventy-three % (eight of 11) of patients with E-rosctte positive lymphoblasts (T ALL) had strong focal paranuclear acid phosphatase (AcP) activity in more than 75% of their lymphoblasts; lymphoblasts from only 6% (three of 48) (p = 0.005) of patients with E-rosette negative, surface immunoglobulin negative lymphoblasts (non-T, non-B ALL) exhibited this characteristic AcP activity. The non-T, non-B ALL cases that manifested focal paranuclear AcP activity had clinical features more characteristic of the T ALL cases. The distribution of β-glucuronidase activity in the lymphoblast cytoplasm was similar to that of AcP for T ALL and non-T, non-B ALL but the stain was generally more difficult to interpret than the AcP and was a less reliable indicator of immunologic type of ALL. The periodic acid-Schiff (PAS) and nonspecific esterase stains were not useful in distinguishing between T ALL and non-T, non-B ALL but PAS negativity was associated with certain clinical features within the non-T, non-B ALL group. Cytochemical evaluation of the lymphoblasts at diagnosis in patients with ALL may be useful in identifying subsets of ALL that have distinct immunologic and clinical characteristics and important therapeutic and prognostic implications.