The gene for adenomatous polyposis coli has been localized to 5q21-22. We have mapped six probes from this region using isotopic or nonisotopic in situ hybridization. Using tritium-labeled probes we localized II227 (D5S37) to 5q14-15 and ECB27 (D5S98) to 5q21. Following hybridization with biotin-labeled probes, the positions of signals along the chromosomes were measured as fractional length relative to the length of the chromosome arm from centromere to qter (FLcen-qter). Ninety-five percent confidence limits, compared with standard karyotypes, provided the corresponding band localization. By this method we localized Cllpll (D5S71) to FLcen-qter 0.407-0.452 (5q21.1-21.3), ECB27 to FLcen-qter 0.426-0.473 (5q21.3), YN5.48 (D5S81) to FLcen-qter 0.459-0.496 (5q21.3-22.2), and ECB134 (D5S97) to FLcen-qter 0.509-0.533 (5q22.3-23.1). ECB220 had three sites of hybridization, a major site at FLcen-qter 0.460-0.492 (5q21.3-22.1) and minor sites at FLcen-qter 0.299-0.339 (5q14.3-15) and FLcen-qter 0.629-0.691 (5q23.3-31.2). We have shown that the chromosome 5 breakpoint in a t(5;15) translocation from a patient with Gardner's syndrome (GM03314) is between Cllpll and ECB27. Linkage data are presented suggesting that ECB27 is located on the same side of the APC locus as II227. These and published results including data on several constitutional deletions (M, SD, and brothers PW and ND) give a probable order of [cen] - [II227, proximal SD breakpoint] - [Cllpll] - [proximal PW/ND, M breakpoint(s), GM03314 breakpoint] - [ECB27] - [APC] - [YN5.48] - [distal PW/ND breakpoint] - [ECB134] - [distal M breakpoint] - [qter]. The major site of ECB220 appears to be between ECB27 and the distal PW/ND breakpoint; the distal SD breakpoint is distal to YN5.48.