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The ancient Maya of the Classic period (1700-1050 B.P.) relied on maize agriculture to support their populations. The agricultural systems they employed to produce that maize varied in form and degree of intensity, with more productive forms of agriculture needed in the Late Classic period (1350-1050 B.P.) to sustain the peaking population. It is likely that the ancient systems of production agriculture contributed to environmental degradation that in turn contributed to the pressures that culminated in the collapse of the civilization. In this study, stable carbon isotope ratios contained in the soil organic matter were used to further investigate ancient maize cultivation in the Petexbatan region of Guatemala. Maize, a plant that uses the C4 photosynthetic pathway, leaves a different carbon (13C) isotopic signal in the soil than the C3 plants of the native forest vegetation. Soil profiles were collected from various landscape features around the Classic Maya site of Aguateca: bajos (or wetlands), control locations (areas not conducive to agriculture), defensible locations (areas within or near defensive walls), rehoyadas (natural karst depressions), and upland locations (level soils from across the rest of the landscape). The samples were tested for various chemical and physical properties, and the bulk soil organic matter and humin fractions were analyzed on a mass spectrometer to determine δ13C values. Graphs of the isotopic values were examined and the bulk and humin δ13C enrichment values for the landscape feature categories were compared statistically using ANOVA methods. We determined that the bulk and humin δ13C values of the Bajo and Rehoyada categories showed significantly greater enrichment than the Control, Defensible, and Upland locations. This enrichment was likely the result of sustained periods of maize cultivation, especially in the Rehoyadas. Surface soil bulk and humin δ13C enrichment values do not show that the Bajo or Rehoyada categories are influenced by modern C4 vegetation, nor do the enrichment values of the Control, Defensible or Upland soils. In other words, C3 vegetation dominates each of the landscape features today under natural conditions and probably would have done so anciently absent human interference. (Savanna soils are an exception, but none were identified in the study area.) When the thinner midslope (Backslope) Rehoyada profiles were compared to Control, Defensive, and Upland samples of similar depth, the Backslope Rehoyada profiles had significantly greater bulk δ13C enrichment values that may have resulted from maize cultivation. There was also a significant difference among the surface soil bulk and humin δ13C values of the Backslope Rehoyada, Control, Defensive, and Upland categories, though the reasons are less clear. Both the Bajos and Rehoyadas would have been valuable agricultural resources for maize production for the ancient Maya.