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Objectives: To compare in vitro physical and mechanical characteristics of 1-layer and 2-layer end-to-end jejunoileostomy., Study Design: In vitro experimental study., Animals: Adult horses (n = 6)., Methods: Harvested equine jejunum and ileum was used to create 1- and 2-layer end-to-end jejunoileostomy specimens. Construction time, bursting pressure, and relative lumen diameter (anastomosis diameter expressed as a percentage of the lumen diameter of adjacent jejunum and ileum) were compared. Construction time and relative lumen diameters were compared using a paired t-test. Bursting pressure for anastomoses and control jejunal segments were compared using a repeated-measure ANOVA. Statistical significance was set at P < .05., Results: Mean (± SEM) construct completion times were shorter for 1 layer (21 ± 0.91 minutes) than 2 layers (26.71 ± 1.16 minutes; P = .005). Relative lumen diameters (percentage of jejunal diameter) were larger for 1 layer (77.67 ± 4.46%) than for 2 layers (69.37 ± 2.8%; P = .035). There were no significant differences in bursting pressures between the 2 groups and the control jejunum (P =.155) or relative lumen diameters (percentage of ileal diameter; P =.118)., Conclusions: One-layer jejunoileostomy can be created in a shorter time and maintain a larger anastomosis luminal diameter without compromising maximum bursting pressure when compared to 2-layer jejunoileostomy., (Copyright 2012 by The American College of Veterinary Surgeons.)