Although vortex pinning in laser-ablated YBa2Cu3O7-δ films on (100) SrTiO3 is dominated by threading dislocations (Dam B et al (1999) Nature 399 439), many other natural pinning sites are present. To identify the contribution from twin planes, surface corrugations and point defects, we manipulate the relative densities of all defects by post-annealing films with various as-grown dislocation densities, ndisl. While a universal magnetic field B dependence of the transport current density js (B, T) is observed (independently of ndisl, temperature T and the annealing treatment), the defect structure changes considerably. Correlating the microstructure to js (B, T), it becomes clear that surface roughness, twins and point defects are not important at low magnetic fields compared to linear defect pinning. Transmission electron microscopy indicates that threading dislocations are not part of grain boundaries nor are they related to the twin domain structure. We conclude that js (B, T) is essentially determined by pinning along threading dislocations, naturally induced during the growth process. Even in high magnetic fields, where the vortex density outnumbers ndisl, it appears that linear defects stabilize the vortex lattice by means of the vortex-vortex interaction.