Paleoecological and geomorphological studies indicate that, during the middle Holocene, there was a predominance of drier conditions with grassy savannahs replacing forests across the South American continent. Modern savannahs are composed mainly of C4 plants and soils developed under this type of vegetation show enrichment in ¹³C compared to soils under C3 vegetation cover. If soils contain stabilized organic matter formed in the middle Holocene, we hypothesize that former C4 vegetation would be evidenced by a large enrichment of ¹³C in soil organic matter (SOM). We investigate this possibility examining the depth variation of carbon isotopic composition in 21 soil profiles collected by different researchers at 14 different sites in Brazil. Of these, profiles from only three sites showed a marked increase of ¹³C with depth (9-10‰ enrichment in δ¹³C difference between the surface soil and deepest depth); two sites showed intermediate enrichment (4-5‰), and nine sites showed a small enrichment of approximatelly 2.5‰. The majority of sites showing all-C3 derived SOM were in the Amazon region. Possible causes for the absence of a large ¹³C enrichment with depth are: (1) dominance of C3 rather than C4 grasses in mid-Holocene savannahas, (2) soil profiles did not preserve organic matter derived from mid-Holocene plants, (3) the retreat of forest areas did not occur on a regional scale, but was a much more localized phenomenon.