Many ascomycete fungi spurt their spores from asci pressurized by osmosis. This paper explores the details of this process in the coprophilous species Ascobolus immersus, through a combination of biomechanical and biochemical experiments, and mathematical modeling. A. immersus forms large asci that expel 8 spores as a single, mucilage-embedded projectile. Measurements of ascus turgor using a microprobe attached to a strain gauge showed a pressure of 0.3 MPa or 3 atm. Analysis of ascus sap using GC/MS identified glycerol as a major osmolyte, accounting for 0.1 MPa of the osmotic pressure within the ascus sap. A mathematical model indicated that a pressure of 0.2 MPa would be sufficient to propel the cluster of ascospores over the distance measured in previous studies. The difference between the measured and predicted pressures is ascribed to loss of pressure as the spores are forced through the tip of the open ascus.