INTRODUCTION AND HYPOTHESIS This study describes a technique to quantify muscle fascicle directions in the levator ani (LA) and tests the null hypothesis that the in vivo fascicle directions for each LA subdivision subtend the same parasagittal angle relative to a horizontal reference axis. METHODS Visible muscle fascicle direction in the each of the three LA muscle subdivisions, the pubovisceral (PVM; synonymous with pubococcygeal), puborectal (PRM), and iliococcygeal (ICM) muscles, as well as the external anal sphincter (EAS), were measured on 3-T sagittal MRI images in a convenience sample of 14 healthy women in whom muscle fascicles were visible. Mean ± standard deviation (SD) angle values relative to the horizontal were calculated for each muscle subdivision. Repeated measures ANOVA and post-hoc paired t tests were used to compare muscle groups. RESULTS Pubovisceral muscle fiber inclination was 41 ± 8.0°, PRM was -19 ± 10.1°, ICM was 33 ± 8.8°, and EAS was -43 ± 6.4°. These fascicle directions were statistically different (p < 0.001). Pairwise comparisons among levator subdivisions showed angle differences of 60° between PVM and PRM, and 52° between ICM and PRM. An 84° difference existed between PVM and EAS. The smallest angle difference between levator divisions was between PVM and ICM 8°. The difference between PRM and EAS was 24°. All pairwise comparisons were significant (p < 0.001). CONCLUSIONS The null hypothesis that muscle fascicle inclinations are similar in the three subdivisions of the levator ani and the external anal sphincter was rejected. The largest difference in levator subdivision inclination, 60°, was found between the PVM and PRM.