Chromosomal translocations involving band 3q27 with various different partner chromosomes represent a recurrent cytogenetic abnormality in B-cell non-Hodgkin’s lymphoma. In a fraction of these translocations, the chromosomal breakpoint is located within the 5' noncoding region of the BCL-6 proto-oncogene where the BCL-6 major breakpoint region (MBR) maps. As a result of the translocation, BCL-6 expression is deregulated by promoter substitution. However, between 30 and 50% of lymphomas with cytogenetically detectable translocations affecting band 3q27 retain a germ-line configuration at the BCL-6 locus. To identify possible additional breakpoint clusters within 3q27, we cloned a t(3;14)(q27;q32) lymphoma without MBR rearrangement and found a novel breakpoint site located between 245 and 285 kb 5' to BCL-6. Breakpoints within this newly described region, which we called the alternative breakpoint region (ABR), were found to be recurrent in lymphomas carrying t(3q27) chromosomal translocations but devoid of BCL-6 MBR rearrangements. Comparative analysis of multiple lymphomas carrying rearrangements within the ABR showed that the breakpoints cluster within a 20-kb distance. Translocations involving the ABR may juxtapose BCL-6 to distantly acting, heterologous transcriptional regulatory elements which cause deregulation of the proto-oncogene. The identification of BCL-6 ABR provides new tools for the diagnosis of lymphomas carrying aberrations at 3q27 and deregulated BCL-6 genes.