Abstract In contrast to other acid phosphatases, four cytoplasmic isoforms (AP1, AP2, AP3A and AP3B) purified from mature soybean seeds presented high activities at temperatures above 80°C, when p-nitrophenylphosphate (p-NPP) was utilized as substrate. However, with tyrosine phosphate and inorganic pyrophosphate as substrates, maximum activities were observed at temperature of 60°C during 10 min reaction. In the absence of substrate, enzymes lost only 20% activity after 60 min at 60°C; the isoforms AP3A and AP3B retained 30% of activity at 70°C after 60 min and all the isoforms were inactivated at 80°C, after 5 min. Thermal inactivation studies indicated that the soybean enzymes showed different temperature dependences in relation to most plant acid phosphatases. A best protective effect was observed when the isoforms were preincubated, at 70°C, with phosphate (10 mM) and p-nitrophenol (10 mM) which indicates that the enzyme inactivation was prevented only in the presence of both reaction products.