We sought polypeptide markers of pathways of lymphoid differentiation by comparing the protein-synthesis patterns of a pair of autologous human lymphoblastoid cell lines: the B-cell line CCRF-SB and the T-cell line CCRF-HSB-2. Cells were cultured in standard medium for 24 or 120 h, then biosynthetically radiolabeled with [3H]leucine. Whole cell lysates were subjected to two-dimensional gel electrophoresis and patterns of protein synthesis were made visible by fluorography. Replicate cultures showed highly reproducible patterns, but differing culture durations produced alterations in the relative synthetic rates of some proteins. Protein synthetic patterns were analyzed visually and with the GELLAB system for computer analysis of two-dimensional gel images. Qualitative and quantitative differences were found between the T- and B-cell patterns. GELLAB detected and accurately paired most of the well-defined spots in each image and constructed a unified data base from data on our initial gels. Statistical analysis of this data revealed several marker proteins, the relative synthetic rates serving to distinguish the T-cell line from the B-cell line. Such computerized analysis of protein synthetic patterns of malignant cells appears likely ultimately to provide important diagnostic information regarding human malignancies.