Pericarp discs were excised from mature green and red ripe tomato (Lycopersicon esculentum Mill. cv. Jackpot) fruit and kept in sterile tissue culture plates for 4 days, including 2 days of incubation with D-[U-13C]-glucose. Cell walls were prepared and differentially extracted with dimethylsulfoxide (DMSO), trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA). Na2CO3, 4 M KOH and 8 M KOH. Cell wall noncellulosic neutral sugar (NS) composition and cell wall synthetic capacity (i.e. incorporation of density label into cell wall sugars) were determined by using a gas chromatograph coupled to a flame ionization detector and a mass spectrometer, respectively. In the crude cell wall, there was significantly less galactose (Gal) and glucose (Glc) in the “outer”2-mm pericarp region, including the cuticle, compared to the “inner”2-mm region immediately below it (closer to the locules). In the CDTA-soluble pectin, rhamnose (Rha), arabinose (Ara) and Gal accounted for approximately 90% of the total NS. The ratios of these sugars were very similar in the total (12C plus 13C) sugars, and also in the newly synthesized ([13C]-labeled) sugars, suggesting that newly synthesized NS associated with the chelator-extractable pectic fraction has a composition very similar to that of preexisting NS. In the 4 M KOH-soluble material, xylose (Xyl) and Glc accounted for approximately 70% of the total NS. The ratio of these sugars was very similar in the total sugars, but much lower in the newly synthesized portion. This suggests that the hemicellulosic polymers synthesized during the ripening process are different in type and/or proportion from those present in the developing fruit. Because the outer pericarp of tomatoes contains at least two distinct tissue types and these have a distinct cell wall composition, analysis of tomato cell wall polysaccharide composition by homogenization of the entire outer pericarp will obscure subtle changes associated with ripening/softening within specific tissue types.