Abstract: Objective To collect the normal spermatozoal gene expression sequence tags with the restriction display technique for constructing a microarray to understand spermatozoal gene expression profiles. Methods The total RNA extracted from normal human spermatozoa were reversely transcribed into cDNAs, which were digested by Sau3AI and linked to universal adapters (adapter 1) at both ends. According to the sequence of the adapter, a pair of primers (universal primers 1) was designed, followed by PCR with primers 1 and the PCR products were transferred into E.coli. The positive clones were collected after identification to serve as the probes for constructing the gene expression microarray of spermatozoa by printing those probes on the slides. The accomplished microarrays were examined by Cy3-labeled normal spermatozoal samples. Results Altogether 1 859 probes were collected, from which 368 were picked out randomly for constructing the microarray. Conclusions Human spermatozoa contain a rich repertoire of RNAs, and the probes we prepared possess good incredibility and speciality.