GDNF-regulated gene expression was studied in cultures of actively self-renewing spermatogonial stem cells established from 6 day old male mice. GDNF is the essential growth factor regulating mouse spermatogonial stem cell self-renewal. Using a serum-free chemically defined culture system that supports mouse spermatogonial stem cell self-renewal for extended periods of time, GDNF-regulated genes were identified using microarray profiling. Keywords: GDNF withdrawal and time-course replacement Overall design: Established cultures of highly enriched self-renewing spermatogonial stem cells were subjeted to withrawal of GDNF and GFRalpha1 for 18-hr followed by replacement of the growth factors for 2, 4, and 8-hr. Gene expression was studied using microarray profiling prior to withdrawal, after withdrawal and at each time-point of GDNF/GFRalpha1 replacement.