Domain 10 of type III fibronectin (10FNIII) is known to play a pivotal role in the mechanical interactions between cell surface integrins and the extracellular matrix. Recent molecular dynamics simulations have predicted that 10FNIII, when exposed to a stretching force, unfolds along two pathways, each with a distinct, mechanically stable intermediate. Here, we use single-molecule force spectroscopy combined with protein engineering to test these predictions by probing the mechanical unfolding pathway of 10FNIII. Stretching single polyproteins containing the 10FNIII module resulted in sawtooth patterns where 10FNIII was seen unfolding in two consecutive steps. The native state unfolded at 100(+/-20) pN, elongating (10)FNIII by 12(+/-2) nm and reaching a clearly marked intermediate that unfolded at 50(+/-20) pN. Unfolding of the intermediate completed the elongation of the molecule by extending another 19(+/-2) nm. Site-directed mutagenesis of residues in the A and B beta-strands (E9P and L19P) resulted in sawtooth patterns with all-or-none unfolding events that elongated the molecule by 19(+/-2) nm. In contrast, mutating residues in the G beta-strand gave results that were dependent on amino acid position. The mutation I88P in the middle of the G beta-strand resulted in native like unfolding sawtooth patterns showing an intact intermediate state. The mutation Y92P, which is near the end of G beta-strand, produced sawtooth patterns with all-or-none unfolding events that lengthened the molecule by 17(+/-2) nm. These results are consistent with the view that 10FNIII can unfold in two different ways. Along one pathway, the detachment of the A and B beta-strands from the body of the folded module constitute the first unfolding event, followed by the unfolding of the remaining beta-sandwich structure. Along the second pathway, the detachment of the G beta-strands is involved in the first unfolding event. These results are in excellent agreement with the sequence of events predicted by molecular dynamics simulations of the 10FNIII module.