Purpose: This study evaluated the effect of different screw-tightening sequences, forces, and methods on the stresses generated on a well-fitting internal-connection implant (Astra Tech) superstructure. Materials and Methods: A metal framework directly connected to four parallel implants was fabricated on a fully edentulous mandibular resin model. Six stone casts with four implant replicas were made from a pickup impression of the superstructure to represent a "well-fitting" situation. Stresses generated by four screw-tightening sequences (1-2-3-4, 4-3-2-1, 2-4-3-1, and 2-3-1-4), two forces (10 and 20 Ncm), and two methods (one-step and two-step) were evaluated. In the two-step method, screws were tightened to the initial torque (10 Ncm) in a predetermined screw-tightening sequence and then to the final torque (20 Ncm) in the same sequence. Stresses were recorded twice by three strain gauges attached to the framework (superior face midway between abutments). Deformation data were analyzed using multiple analysis of variance at a .05 level of statistical significance. Results: In all stone casts, stresses were produced by the superstructure connection, regardless of screw-tightening sequence, force, and method. No statistically significant differences for superstructure preload stresses were found based on screw-tightening sequences (-180.0 to -181.6 µm/m) or forces (-163.4 and -169.2 µm/m) (P > .05). However, different screw-tightening methods induced different stresses on the superstructure. The two-step screw-tightening method (-180.1 µm/m) produced significantly higher stress than the one-step method (-169.2 µm/m) (P = .0457). Conclusions: Within the limitations of this in vitro study, screw-tightening sequence and force were not critical factors in the stress generated on a well-fitting internal-connection implant superstructure. The stress caused by the two-step method was greater than that produced using the one-step method. Further studies are needed to evaluate the effect of screw-tightening techniques on preload stress in various different clinical situations. Keywords: internal-connection dental implant, preload stress, screw-tightening sequence, tightening force, tightening method