Before fertilization can occur, ejaculated mammalian spermatozoa must undergo a maturation process known as capacitation, which is dominated by post-translational modifications, particularly phosphorylation. Despite its biological importance, characterization of those proteins targeted for phosphorylation during capacitation remains ill-defined. Here, we report the isolation and purification of 288 phosphorylated peptides from rat spermatozoa using titanium dioxide columns in combination with nanoflow mass spectrometry. This equated to 120 identified phosphorylated proteins present in pure populations of spermatozoa. The MS survey scans of replicate titanium dioxide eluates, derived from both noncapacitated and capacitated sperm lysates, were then compared in silico using a virtual 2D PAGE format and DeCyderMS software. This analysis found 15 differentially phosphorylated proteins during capacitation. Included in this list were sperm qualifiers such as Izumo, a known sperm-oocyte fusion protein. To demonstrate that this label-free quantitative approach to phosphoprotein analysis was viable, we measured the enzymatic activity of 5′-nucleotidase, the phosphorylation status of which changed during capacitation. The results revealed, for the first time, that 5′-nucleotidase activity is up-regulated as sperm capacitate. This change, together with the other protein identifications reported in this study, constitute important new leads in elucidating the biochemical mechanisms by which spermatozoa attain a capacitated state. © 2010 American Chemical Society.