A Noti-linking clone NL1-210 (D3S1656) that contains the human MAP kinase activated protein kinase (3PK) gene was localized to 3p21.2 on DAPI-banded and propidium iodide (R-bands)-stained chromosomes by fluorescence in situ hybridization (FISH). For more precise localization of 3PK, two cosmid probes were used as a frame. In order to establish this frame, two Noti-linking clones, NL2-008 (D3S1648) and NL3-003 (D3S3872) were used to screen the cosmid library for locus extension. They mapped to 3p21 and were found to belong to two separate contigs of Noti-jumping and linking clones. Using FISH on DAPI-banded metaphase chromosomes, we have determined the precise localization of cosNL2-008 and cosNL3-003 to 3p21.2-p21.1 and 3p22-p21.3 respectively. The 3PK gene was localized to the 3p21.2 region within this frame by two-colour FISH. The orientation of the probes are tel-D3S3872-3PK-D3S1648-cen.