The inability of HT-29 cells to undergo an enterocytic differentiation when grown in a glucose-containing (Glc+) medium has been recently correlated to an overall impairment of N-glycan processing. These results were obtained using confluent HT-29 cells in which the differentiation characteristics are fully expressed under differentiation permissive conditions (glucose-deprived medium, Glc-). Whether these changes of N-glycan processing appear progressively during the cell growth or are already present from the beginning of the culture was investigated in this work by comparing the actual status of N-glycan processing in both exponentially growing Glc+ and Glc- HT-29 cells. Under these conditions, HT-29 cells do not express any characteristics of enterocytic differentiation, even when grown in differentiation permissive conditions. We show here that the conversion of high-mannose to complex glycoproteins is, however, severely reduced in HT-29 cells grown in differentiation non-permissive conditions (HT-29 Glc+) whatever the phase of growth studied. In contrast, HT-29 cells grown in differentiation permissive conditions (HT-29 Glc-) display a normal pattern of N-glycan processing in both the exponential and the stationary phase of growth. We also show that both growing and confluent HT-29 Glc+ cells accumulate Man GlcNAc2 species, thus suggesting that there is an important regulatory point at this level. We therefore conclude that the N-glycan processing may be used as an early biochemical probe for the enterocytic differentiation of HT-29 cells. Whether these early changes result from an early metabolic regulation or are the consequence of a genetic control remains to be studied.