A-scan readings must be taken along the visual axis for accurate axial length measurement. Various fixation targets, which ideally should not induce accommodation, are used to limit eye movements. In this study results using the Humphrey biometer under different fixation conditions were compared to establish the most suitable target. We also investigated corneal applanation effects by comparing optical and ultrasonic measurements of anterior chamber depth. We examined 12 randomly selected normal eyes of 12 young subjects. Three A-scans were recorded for each of five fixation conditions: spotlight at 6 m; Snellen letter at 6 m; solid probe's built-in green fixation target; Snellen letter at 6 m viewed through a -4.00 DS lens; spotlight at 6 m under cycloplegia (the comparison condition). Variability 'within' each fixation condition, estimated by the standard deviation for each subject, was equal throughout the 12 subjects for each fixation condition. Also variation was independent of size of the ocular dimension. The good agreement between the comparison condition and optical anterior chamber depth measurements indicated that the solid probe produced little applanation. Differences 'between' fixation conditions were investigated. It was concluded that a spotlight or letter at 6 m provides a suitable fixation target for biometry. Built-in fixation targets can produce significant errors.