This study investigated factors that influence occurrence and persistence of plant DNA in the soil environment in three crop rotations. In each rotation, soil was sampled in May before planting, in July and August while crops were growing, and in October after harvest. Total DNA was recovered from soil samples taken at two different depths in the soil profile and quantified. Three target plant genes (corn CP4 epsps, corn 10-kD Zein, and soybean CP4 epsps) also were quantified in these DNA extracts using species-specific quantitative real-time PCR assays. In general, total plant DNA content in the soil environment was greatest when the crop was growing in the field and decreased rapidly after harvest. Nevertheless, low levels of target plant DNA were often still detectable the following spring. Age of rotation did not influence target DNA quantities found in the soil environment. Data were collected for a combination of 10 location-years, which allowed for estimation of the variance components for six factors including time of sampling, year, location, crop, sampling depth, and herbicide to total and target DNA content in the soil samples. Mean target recombinant DNA content in soil was influenced most strongly by time of sampling and year (85 and 6%, respectively), whereas total soil DNA content was less dynamic and was most strongly influenced by location and year (49 and 25%, respectively). Over the duration of this study, no accumulation of transgenic plant DNA in the soil environment was observed.