Using the human/hamster cell line UCTP2A-3, we have generated and isolated three hybrids, each containing a novel human chromosome 3p break. All chromosome 3 materials distal to the breaks were lost. Two of the breakpoints were produced using aphidicolin induction; the third breakpoint occurred spontaneously. The aphidicolin-induced breaks were localized to 3p21.1 in hybrid AR1 and to p14.1 in hybrid AR2. The spontaneous break was localized to 3p11 in hybrid 2A-3-1. These hybrids were used to sublocalize 18 chromosome 3 probes further to five regions within 3cen-p21.1. The new hybrids and sublocalized markers will be useful in the study and characterization of the 3p11, 3p14.1, and 3p21.1 segments of chromosome 3.