BACKGROUND Perception of upright is often assessed by aligning a luminous line to the subjective visual vertical (SVV). OBJECTIVE Here we investigated the effects of visual line rotation and viewing eye on SVV responses and whether there was any change with head tilt. METHODS SVV was measured using a forced-choice paradigm and by combining the following conditions in 22 healthy subjects: head position (20° left tilt, upright and 20° right tilt), viewing eye (left eye, both eyes and right eye) and direction of visual line rotation (clockwise [CW] and counter clockwise [CCW]). RESULTS The accuracy and precision of SVV responses were not different between the viewing eye conditions in all head positions (P > 0.05, Kruskal-Wallis test). The accuracy of SVV responses was different between the CW and CCW line rotations (p ≈ 0.0001; Kruskal-Wallis test) and SVV was tilted in the same direction as the line rotation. This effect of line rotation was however not consistent across head tilts and was only present in the upright and right tilt head positions. The accuracy of SVV responses showed a higher variability among subjects in the left head tilt position with no significant difference between the CW and CCW line rotations (P > 0.05; post-hoc Dunn’s test). CONCLUSIONS In spite of the challenges to the estimate of upright with head tilt, normal subjects did remarkably well irrespective of the viewing eye. The physiological significance of the asymmetry in the effect of line rotation between the head tilt positions is unclear but it suggests a lateralizing effect of head tilt on the visual perception of upright.