Objective To create a rapid, inexpensive, efficient, and reproducible real-time three-dimensional (3-D) analysis of viable spermatozoa. Previous studies have demonstrated that abnormal semen profiles are associated with a modest increase in the frequency of sperm chromosomal abnormalities, and that sperm with aberrations in the shape and contours of the head may be carriers of chromatinic defects. Although high-power magnification and enhanced video-generated magnification have been suggested, these techniques are inherently limited by the clarity of the image, the time required for the analysis, and the risk of variable head-positioning during imaging. Design In vitro experiment. Setting University-affiliated infertility research laboratory. Patient(s) Anonymous sperm donors. Intervention(s) Individual motile sperm were identified, analyzed at ×600 magnification, and a 10-second digital video was obtained. Main Outcome Measure(s) Image-tracking software captured serial photographs of sperm from recorded videos. Images were automatically extracted from each video frame using enhanced correlation coefficient maximization; the general shape of the sperm was extracted via space-carving. The reconstructed image was rotated to permit viewing from any direction, and the final image was rendered through interpolation. Result(s) This technique yielded images that enable noninvasive, 3-D, real-time, in vitro assessment of sperm surface morphology. Conclusion(s) This proof-of-principle demonstrates that by keeping spermatozoa in a fluid environment, a 3-D sperm–surface reconstruction can be created. This technique can be automated, requires minimal computing power, and utilizes equipment already available in most embryology laboratories.