Purpose: To determine whether intracameral acetylcholine can contract pupils dilated with intracameral mydriatics in phacoemulsification cataract surgery. Methods: A total of 60 patients were included in this prospective randomized masked study performed at ornskoldsviks Hospital Eye Clinic. The patients were randomized and were given either topical placebo and an intracameral mydriatic solution (ICM) (cyclopentolate 0.1%, phenylephrine 1.5% and xylocaine 1%) (n=30) or topical mydriatics (TM) (cyclopentolate 0.85% and phenylephrine 1.5%) and xylocaine 1% intracamerally (n=30) at the start of surgery. After intraocular lens (IOL) implantation, 0.15ml 1% acetylcholine was given intracamerally in all cases. The pupil size was registered preoperatively, 45seconds after intracameral injection, after ophthalmic viscosurgical device (OVD) evacuation, 30seconds after acetylcholine injection, 2min after acetylcholine injection and the day after surgery. Results: The pupil contraction and pupil size after acetylcholine injection showed no significant differences at 30seconds (contraction 1.0 +/- 0.4 in ICM group versus 0.9 +/- 0.4 in TM group; p=0.75; size 4.8 +/- 1.1 in the ICM group versus 5.2 +/- 1.1 in the TM group; p=0.24) or at 2min (contraction 1.5 +/- 0.6 in the ICM group versus 1.4 +/- 0.6 in the TM group; p=0.63; size 4.3 +/- 0.9 in the ICM group versus 4.7 +/- 1.0 in the TM group; p=0.13). No difference in baseline pupil size after ophthalmic viscosurgical device (OVD) evacuation was seen between the two groups (5.8 +/- 0.9 in the ICM group versus 6.1 +/- 1.2 in the TM group; p=0.28). Conclusion: We here show that intracameral acetylcholine contracts pupils as effectively after dilatation with intracameral mydriatics as after dilatation with topical mydriatics. Cataract surgeons can feel comfortable and safe when using intracameral mydriatics, even if pupil contraction with acetylcholine should be required.