Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.