Two distinct alpha-(1-3)-fucosyltransferase activities have been identified in the colon carcinoma cell lines HT-29 and COLO-205. While both enzymatic activities exhibit similar affinities for a synthetic alpha-(1-3) acceptor and GDP-fucose, they differ with respect to divalent cation requirements, N-ethylmaleimide inhibition, and glycoprotein substrate specificity. The COLO-205 alpha-(1-3) activity exhibits maximal enzymatic activity in the presence of 20 mM Mn2+ but retains less than 10% activity in the absence of divalent cations. In contrast, the optimal Mn2+ concentration for the HT-29 enzyme is 1 mM, although this activity is relatively insensitive to divalent cation stimulation. In addition, the HT-29 alpha-(1-3)-fucosyltransferase activity is resistant to inhibition by 30 mM N-ethylmaleimide and relatively inactive toward the glycoprotein substrate fetuin as compared to its desialylated derivative, asialofetuin. The COLO-205 activity is inhibited approximately 90% by N-ethylmaleimide and is equally active with either glycoprotein acceptor. Although the alpha-(1-3) specific activities are similar in both cell lines, N-ethylmaleimide-sensitive alpha-(1-4) fucosyltransferase activity is 40-fold higher in COLO-205 as compared to HT-29, suggesting that the COLO-205 fucosyltransferase activity may be an alpha-(1-3/4) enzyme, while the HT-29 activity appears to be an alpha-(1-3) specific form. Further examination of a panel of cell lines, tumor biopsies, and xenografts, based on the effect of metal ions and N-ethylmaleimide, indicated that both enzyme activities are similarly expressed in human colon carcinoma tissue.