Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcel-lular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several diva-lent cations were determined. SMase-C and other unidentified esterase activity were detected in solubleand particulate fractions. SMase-C was 94.5–96.0% higher than the unidentified esterase activity. Solubleand insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble andinsoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg2+, Mn2+, or Co2+,and inhibited by Zn2+, Hg2+, Ca2+, and EDTA. SMase-C was active in the pH range of 3–10 and its maxi-mum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemicalproperties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had notbeen described before and might be essential for E. histolytica metabolism or virulence.