The purpose of these experiments was to determine if an extracellular calcium binding site gradient is evident in freshly dissected or cultured mouse vibrissa and human scalp follicles and to measure possible drug effects on this gradient. Mouse vibrissae were cultured with or without either minoxidil or pinacidil, and human scalp follicles were cultured with or without epidermal growth factor. Anagen vibrissa and scalp follicles were dissected and placed in culture for 4 h to 4 days, then fixed in a solution containing lanthanum chloride and prepared for either quantitative energy-dispersive X-ray microanalysis (X-ray) or qualitative transmission electron microscopy (TEM). Since lanthanum has a high charge density it displaces Ca2+ ions from anionic binding sites. TEM analysis revealed extensive accumulation of electron-dense lanthanum deposits in the intercellular compartment of differentiating cells in the hair shaft and inner root sheath in the apex of the follicular bulb. Sparse lanthanum precipitate was observed in the intercellular space of the proliferative cells at the base of the bulb. This gradient of lanthanum precipitate was evident in both freshly dissected and cultured vibrissa and scalp hair follicles, irrespective of treatment with drugs that grow hair or epidermal growth factor. X-ray microanalysis indicated that percent by weight of lanthanum was markedly higher in the apex compared to the base of the follicular bulb in vibrissa and scalp follicles. These qualitative and quantitative data demonstrate that an extracellular calcium binding site gradient exists in cultured vibrissa and scalp hair follicles, and that this gradient is not significantly affected by hair growth altering drugs including minoxidil or pinacidil, and epidermal growth factor.