Dipyrone is a prodrug which is used mainly for its analgesic and antipyretic effects. After oral intake, dipyrone is rapidly hydrolyzed to its main metabolite, 4-methylaminoantipyrine (4-MAA), from which many other metabolites are produced by enzymatic reactions. Even though it is well known that dipyrone is a prodrug and hydrolyzed non-enzymatically, in most of the studies of dipyrone the prodrug form is tested using in vitro methodologies, which do not represent or predict the actual in vivo activity of dipyrone. In this study, we characterize the hydrolysis kinetics of dipyrone as functions of concentration, temperature, and pH using a HPLC assay. Concentration is an important factor in the hydrolysis of dipyrone. Low concentrations of dipyrone are hydrolyzed more rapidly than are solutions of higher concentrations. At a concentration of 0.1M, which is 140 times, the concentration of the marketed pharmaceutical form, dipyrone is only minimally (10%) hydrolyzed to 4-MAA at 5h. Temperature, as expected, affects the hydrolysis reaction dramatically. We tested three temperatures (4, 21, and 37 degrees C) and found that at body temperature the hydrolysis is significantly faster than at room or at refrigerator temperatures. Compared with more alkaline solutions, the hydrolysis rate of dipyrone increases dramatically in acidic solutions. At low pH (2.5) and at a 0.01 mM concentration, the hydrolysis of dipyrone is completed within almost 30 min, which is the highest rate we observed. Experiments which involve in vitro and/or local application of dipyrone should consider these physicochemical factors and interpret the results accordingly.