The concur-rent use of atomic force microscopy (AFM) and NMR spectroscopy should enable one to link the structural information from the former to chemical information obtained with the latter. In this instance, the self-assembly of type I collagen into segmental long spacing (SLS) collagen crystallites is examined, and the first concrete evidence for the incorporation of the nucleotide ATP into these crystallites is presented. The type 1 collagen monomer contains no phosphorus, unlike the triphosphate group of the ATP, thus making (31)p NMR spectroscopy a highly specific probe for the location and interaction of ATP. The changes in the (31)p NMR spectral attributes upon self- assembly of SLS collaggen crystallites under various conditions are interpreted with relation to the crystallite morphology observed with AFM. The formation of crystallites is found to correlate with decreased mobility of all three P-31 nuclei of ATP, as evinced by appreciable line broadening in the solution-state spectra. This is attributable to ATP incorporation into the crystallite structure. Analysis of the resonance frequencies for each of the P-31 nuclei of ATP indicates that the oc- and gamma-phosphates have decreased electron density relative to the beta-phosphate upon incorporation, implying direct charge-charge binding, with binding probabilities greater for the alpha- and gamma-phosphate groups than for the beta-phosphate group. Three possible scaffolding roles of the ATP are proposed with respect to the structure of the SLS collagen crystallite based upon the evidence presented.