We studied the regulation of the beta-galactoside alpha2,6-sialyltransferase (hST6Gal I) gene during HL-60 cell differentiation induced with dimethyl sulfoxide (DMSO), all transretinoic acid (ATRA), and phorbol myristate acetate (PMA). During HL-60 cell line differentiation, cell surface levels of alpha2,6-sialic acids expression decreased, as measured by flow cytometric analysis using Sambucus nigra agglutinin (SNA). Activities of hST6Gal I and levels of hST6Gal I mRNA dramatically decreased after 1 day of stimulation. Using reverse transcription polymerase chain reaction (PT-PCR), we found the major hST6Gal I mRNA isoform in HL-60 cells contains 5'-untranslated exons Y and Z. These results suggest that the expression of cell surface alpha2,6-sialic acids is controlled at the mRNA level, which is regulated by a promoter located 5'-upstream of exon Y.