Homeobox-containing genes play critical roles in regulating tissue-specific gene expression essential for tissue differentiation, as well as determining the temporal and spatial patterns of development. Recently, a human cardiac homeobox-containing gene, CSX, has been isolated. CSX is abundantly expressed in the human heart from fetal stages, suggesting that CSX plays an important role in human heart formation. In the present study, we have determined the chromosomal localization of CSX by fluorescence in situ hybridization techniques and systemic screening of a yeast artificial chromosome library using polymerase chain reaction. By these methods, CSX was mapped to 5q34 of human chromosome 5 near the boundary of 5q34 and 5q35. In this region, another homeobox-containing gene MSX2, which is expressed in various tissues including the conduction system of the developing heart, has been assigned. Localization of CSX and MSX2 to the same region of the human chromosome suggests that these genes may be coordinately regulated during human heart formation.