We used atomic force microscopy (AFM), which utilizes a novel 3D image-contrast mechanism, to obtain nanometer-resolved, topographic data images of the natural surface structures of untreated bovine sperm cells. Freshly ejaculated, thawed, sonicated, and demembranated bovine sperm were adsorbed passively or by motility from suspension onto a coverglass substrate and directly imaged in normal air and saline environments without damaging the cells. Our AFM images of the surface structures of unfixed sperm imaged in normal air were consistent with previous electron microscope results on frozen or fixed sperm, demonstrating that the accurate preservation of small cellular structures is achievable using greatly simplified AFM sample preparation and imaging environments. Our AFM results also indicate that imaging sperm in physiologic buffer provides more native views of sperm due to the retention of cytoplasmic structures easily disrupted by drying forces. In addition, the AFM images show that numerous nanometer-sized subcellular structures of the sperm head and tail regions could be clearly visualized on rapidly prepared, unfixed, intact cells. Consequently, AFM should be considered a new tool for studying sperm structure abnormalities and monitoring the specific effects of, or damage caused by, various chemical reactants or other treatments on the structures of metabolically active or partially demembranated sperm. AFM is now emerging as an important new structural technique for imaging hydrated cells and organelles and, in addition, has the capabilities to physically ''interrogate'' them with the local probe, (C) 1995 Academic Press, Inc.