Although "chaperone molecules" rich in negatively charged residues (i.e., glutamic and aspartic acid) are known to play important roles in the biomineralization process, the precise mechanism by which type I collagen acquires intrafibrillar mineral via these chaperone molecules remains unknown. This study demonstrates a mechanism of cooperative nucleation in which three key components (collagen, chaperone molecules, and Ca(2+) and PO(4)(3-)) interact simultaneously. The mineralization of collagen under conditions in which collagen was exposed to pAsp, Ca(2+), and PO(4)(3-) simultaneously or pretreated with the chaperone molecule (in this case, poly(aspartic acid)) before any exposure to the mineralizing solution was compared to deduce the mineralization mechanism. Depending on the exact conditions, intrafibrillar mineral formation could be reduced or even eliminated through pretreatment with the chaperone molecule. Through the use of a fluorescently tagged polymer, it was determined that the adsorption of the chaperone molecule to the collagen surface retarded further adsorption of subsequent molecules, explaining the reduced mineralization rate in pretreated samples. This finding is significant because it indicates that chaperone molecules must interact simultaneously with the ions in solution and collagen for biomimetic mineralization to occur and that the rate of mineralization is highly dependent upon the interaction of collagen with its environment.