Specifications are described for high purity 4-nitrophenol, which is suitable for spectrophotometric standardization. Such a reference material is needed in clinical enzymology to establish the proper molar absorptivity of 4-nitrophenol under final reaction conditions, particularly for measuring alkaline phosphatase activity in human serum. Some lots of 4-nitrophenol available commercially met these specifications, but several did not. The latter can be purified to meet the specifications by recrystallization or sublimation. The molar absorptivity of 4-nitrophenol (35 .mu.mol/l) in 10 mmol/l NaOH at 25.degree. C at 401 nm is 18,380 .+-. 90 l .cntdot. mol-1 .cntdot. cm-1.