Thirty-five single-copy and 17 repetitive sequence DNA probes specific for human chromosome 3 were isolated from human chromosome 3-derived genomic libraries. Seven DNA clones, including three that are polymorphic for BglII or MspI, were mapped by in situ hybridization. Four probes were mapped to 3p subregions and 3 were mapped to 3q subregions. Three of the DNA sequences map to regions overlapping a segment of chromosome 3 (3p14-23) frequently deleted in small cell lung cancer cells. By Southern blot analysis on a deletion hybrid panel, we previously mapped 6 of these probes to three distinct chromosome 3 subregions. Our in situ data support these assignments and more precisely determine the localization of each clone to the following regions: D3S34 (3p14-21), D3S35 (3p21), D3S39 (3p21), D3S40 (3p12-13), D3S37 (3q21-23), and D3S36 (3q21). Clone pL84c, a low repeat sequence clone (approximately 30 copies), was mapped to the 3q21-29 subregion. These DNA clones mapped by in situ hybridization can provide useful landmarks for the ordering and localization of other clones.