Multigenicity is one of the features of cancer/testis-associated genes. In the present study we analyzed the number and expression of genes of the SPANX(CTp11) family of cancer/testis-associated genes. Genomic database analysis, next to the four previously described SPANX genes, revealed the presence of a novel gene: SPANXE. Moreover, we detected an allelic variant of SPANXB resulting in one amino acid substitution in the encoded protein: SPANXB'. Most SPANX genes are present on contig NT_011574 located at Xq26.3-Xq27.1. Based on expressed sequence tag databases and RT-PCR analysis three additional novel SPANX sequences were identified, though not represented so far in the human genome sequence. Sequence alignments justify a subdivision of this gene family based on the absence (SPANXA-likes) or presence (SPANXB) of an 18 base pair sequence stretch in the open reading frame. The alignments also reveal an unusually high level (99%) of intron homology. Furthermore, the nucleotide variations in the open reading frame almost all lead to amino acid substitutions. Southern blot and database analyses indicate that SPANX sequences are exclusively present in primates. With RT-PCR analysis on human sperm cell precursors and tumor cell lines most family members could be detected. SPANXB was only found in sperm cell precursors and could not be detected in the tumor cell lines tested. Overall SPANXA was the most frequently expressed SPANX variant in melanoma and glioblastoma cell lines.