Intracellular antigens are of major importance for immunophenotyping of normal leukocytes as well as leukemias and malignant lymphomas. Immunofluorescence microscopic evaluation of cytocentrifuge preparations has remained the preferred technique for detection of intracellular antigens for a long time. Recently, flow cytometric detection of intracellular antigens has been improved by the development of new permeabilization/fixation solutions. We compared four commercially available solutions: FACS Brand Lysing Solution (FACS Brand; Becton Dickinson, San Jose, CA, USA), Fix & Perm cell permeabilization kit (Fix & Perm; An der Grub, Vienna, Austria), OptiLyse B lysing solution (OptiLyse B; Immunotech, Marseille, France), and ORTHO PermeaFix(PermeaFix; Ortho Diagnostic Systems, Raritan, NJ, USA). These solutions were evaluated for the complexity and duration of the intracellular staining procedure, the effects on light scatter patterns, and the staining results for the intracellular antigens terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD3 (CyCD3), myeloperoxidase (MPO), and cytoplasmic immunoglobulin light chains (CylgL). The four methods could easily be introduced in our laboratory and had only minor effect on the light scatter patterns of the tested cell samples. Each of the four tested antigens was detectable with at least one of the four methods. Only the Fix & Perm cell permeabilization kit could be used for reliable detection of all four intracellular antigens. In a large series of 450 BM and PB samples containing various percentages of TdT+ cells, the results of flow cytometric TdT staining with FACS Brand Lysing Solution were highly comparable to the results obtained by immunofluorescence microscopy (P = <0.00001). Our comparative study shows that flow cytometric detection of the intracellular antigens TdT, CyCD3, MPO, and CylgL can now reliably be performed on a routine basis.