The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.