Abstract: A reference solution for calibrating and monitoring blood gas instrumentation that has a shelf line of two years or greater is disclosed. The solution is contained in a closed container having a gas or gas mixture that is inert to the reference solution and comprises an aqueous mixture containing a hemoglobin, a source of bicarbonate ions and an organic buffer. The solution optionally contains a methemoglobin reducing system, reference concentrations of selected electrolytes, a polyphosphate compound and antibiotics. The ampule is subsequently opened and the solution is equilibrated with a gas mixture having components which provide gasses in a physiological range to the solution. The solution is characterized by exhibiting properties similar to fresh blood subsequent to equilibration.