To provide a sensitive and generally applicable method to detect clonal cells in acute lymphoblastic leukemias (ALL), we have designed a new strategy based on the polymerase chain reaction (PCR) amplification of the T cell receptor gamma delta gene rearrangements found in most T and B lineage ALLs. PCR allows rapid sequencing of variable-(diversity)-joining (V-[D]-J) junctions from tumor DNA and construction of anti-junctional oligonucleotides (AJOs) used as probes to detect clonal cells in the same patient. We have defined oligonucleotides suitable for all T cell receptor (TCR) rearrangements involving functional V gamma segments. Oligonucleotides corresponding to preferential TCR delta rearrangements in T and B lineage ALLs were also used. By analysis of the nucleotide sequence of 52 V gamma-V gamma junctions from 30 cases of B and T ALLs, we demonstrate that V-J junctional sequences are clone specific in both lineages and at all stages of differentiation examined despite the frequent presence of the recently described P nucleotides. Experiments performed with TCR gamma delta AJOs on DNA from tumor cells and polyclonal T cells show that AJOs can be used to differentiate clonal cells from polyclonal T cells, distinguish between different T cell clones, and detect residual clonal populations at 10(-4)/10(-5) dilution. AJOs were also used to detect residual disease in samples from patients in clinical and morphological complete remission. Finally, rearrangement patterns were studied by classical Southern analysis in selected cases at both presentation and subsequent relapse showing absence of clonal evolution in most cases. V-(D)-J nucleotide sequences of rearrangements with an identical pattern of rearrangement at presentation and relapse were identical in all cases analyzed. We therefore describe a new, specific, and clinically useful strategy for the detection of minor clonal populations applicable in the majority of cases of ALL.