The 170-kDa or heavy subunit of the galactose binding adhesin of Entamoeba histolytica is seminal in target cell binding and lysis. To determine the existence and complexity of the 170-kDa subunit gene family, hgl, an amebic genomic library in lambda phage was hybridized with DNA fragments from the 5' or 3' ends of hgl1. Termini from three distinct heavy subunit genes were identified including hgl1, hgl2, and a third, unreported gene designated hgl3. The open reading frame of hgl3 was sequenced in its entirety. Non-stringent hybridization of a genomic Southern blot with heavy subunit specific DNA labeled only those bands predicted by hgl1-3. The amino acid sequence of hgl3 was 95.2% identical to hgl1 and 89.4% identical to hgl2. All 97 cysteine residues present in the heavy subunit were conserved in hgl1-3. Analysis of amebic RNA showed that all three heavy subunit genes were expressed in the amebae and that hgl message became less abundant as the amebae entered a stationary growth phase.