The nuclei of human neutrophils typically consist of a linear array of three or four lobes joined by DNA-containing filaments. Terminal lobes are connected to internal lobes via a single filament, while internal lobes have two filaments, each to an adjacent lobe. Some lobes also have appendages of various shapes and sizes. In particular, up to 17% of neutrophil nuclei of healthy women exhibit a drumstick-shaped appendage that contains the inactive X chromosome. This report provides a detailed analysis of the relationship between nuclear morphology and the location of the X and Y chromosomes in human neutrophils. Fluorescent in situ hybridization analysis revealed that the X and the Y chromosomes of male neutrophil nuclei are randomly distributed among nuclear lobes. Similarly, in female neutrophil nuclei with a drumstick appendage, the active X chromosome is also randomly distributed among lobes. In contrast, the inactive X chromosome is preferentially located in a terminal lobe in over 90% nuclei with drumsticks. Within the terminal lobe of nuclei with drumsticks, the inactive X chromosome lies distal to the point of filament attachment in 80% of the nuclei. The inactive X chromosome also exhibits a specific orientation within the drumstick appendage, with over 95% of nuclei having the X centromere located toward the tip of the appendage. Female nuclei without a drumstick appendage also have one of the X chromosomes (presumably the inactive chromosome) preferentially situated in a terminal lobe. Nonrandom distribution of the inactive X chromosome is discussed in the context of a model that considers chromosomes as determinants of neutrophil nuclear morphology.