Growth in culture of squamous head and neck cancer is hampered by microbial contamination, low plating efficiency, and cellular heterogeneity within tumors. Furthermore, clumps of cells must be removed if plating efficiency is to be accurately determined. Isokinetic velocity sedimentation was applied to 44 primary tumor specimens in an effort to minimize these problems. Seven fractions were evaluated for cell number, clump number, cell viability, clonogenic growth, plating efficiency, and microbial overgrowth. Unseparated specimens were simultaneously cultured. Microbial growth was significantly associated with the highest gradient fraction. Clumps were significantly associated with the lowest gradient fraction. Colony formation was significantly associated with middle gradient isokinetic velocity sedimentation, although seven specimens grew only when fractionated, suggesting the possibility of inhibitor cells within the tumor specimen.