The Drosophila melanogaster flightless I gene is involved in cellularization processes in early embryogenesis and in the structural organization of indirect flight muscle. The encoded protein contains a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat protein-protein interaction domain. We have cloned Fliih, the corresponding chromosomal gene from the mouse, and determined its nucleotide sequence (15.6 kb). The predicted Fliih protein of 1271 amino acids is 95% identical to the human FLII protein. Like the human gene, Fliih has 29 introns, compared with 13 in C. elegans and 3 in D. melanogaster. Fluorescence in situ hybridization was used to map Fliih to Chromosome 11B. Fliih lies adjacent to Llglh, the mouse homologue of the D. melanogaster tumor suppressor gene lethal(2) giant larvae. The sequence of the genomic DNA in this area, combined with cDNA sequences, establishes that the 3' ends of the Fliih and Llglh transcripts overlap. The overlap region contains polyA signals for both genes and is conserved between human and mouse.