We recently reported the isolation of two new members of the sulfatase gene family, arylsulfatase D (ARSD) and E (ARSE), located approximately 50 kb from each other in the Xp22.3 region. Mutation analysis indicated ARSE as the gene responsible for X-linked recessive chondrodysplasia punctata. Expression of the ARSE gene in COS cells resulted in a heat-labile arylsulfatase activity that was inhibited by warfarin. At the same time, we detected the presence of a 1.2-kb fragment located at approximately 60 kb from ARSD and ARSE with significant homology to these two genes, suggesting the existence of another sulfatase gene, arylsulfatase F (ARSF), in Xp22.3. We have used a combined approach of long-range genomic sequencing and screening of cDNA libraries to isolate the ARSF gene. Expression of the ARSF cDNA in COS cells resulted in a heat-labile arylsulfatase activity that is not inhibited by warfarin, supporting our hypothesis that only ARSE is specifically inhibited by warfarin and is most likely involved in warfarin embryopathy. Genomic analysis revealed that ARSF has an intron/exon organization highly similar to those of ARSD and ARSE, which is also shared by another Xp22.3 sulfatase gene, ARSC (arylsulfatase C, also known as steroid sulfatase), with the splice sites occurring at the same position in all four genes. The data obtained from sequence analysis and presented in this paper indicate that the ARSC, ARSD, ARSE, and ARSF genes are more similar to each other than to other members of the sulfatase gene family, supporting our hypothesis that they represent a subfamily of related proteins created through duplication events that occurred in an ancestral pseudoautosomal region.