The production of diffraction-quality crystals of Munc18c, a protein involved in regulating vesicular exocytosis in mammals, is reported. The diffraction resolution of Munc18c crystals was optimized by (i) cocrystallizing with a peptide fragment of the Munc18c functional binding partner syntaxin4, (ii) using nanolitre free-interface diffusion crystallization-screening chips and microlitre hanging-drop vapour diffusion and (iii) applying a post-crystallization dehydration treatment. Crystals belonging to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 170.8 A, alpha = beta = gamma = 90 degrees , were generated that diffract to 3.7 A resolution on a laboratory X-ray source.