Because of the accessible and renewable nature of feedstock and the potential for the reduction of harmful combustion emissions and greenhouse gases, biodiesels have received increasing interest as an alternate fuel. Oral exposure to biodiesels is a concern because of contact during refuelling, accidental ingestion and exposure through ground water contamination. Although biodiesels from various feedstock are in use commercially and experimentally, very little is known about their potential adverse effects and no data is available on their potential for ground water contamination. A study was performed on male rats following oral treatment with experimental biodiesels (dissolved in corn oil) derived from canola oil (Bio-C), soy oil (Bio-S) and fish oil (Bio-F), at 500 mg/kg body weight/day, 5 days per week, for 4 weeks. Separate groups of animals were treated with low sulfur diesel (LSD) for comparison purpose, and with corn oil alone to serve as control. The potential for ground water contamination by biodiesels was investigated by the preparation of water-accommodated fractions (WAF) followed by gas chromatographic analysis. WAF from Bio-F and Bio-S was found to have the highest level of dichloromethane extractable materials. Gas chromatographic analysis indicated that the extractable materials from biodiesels contained much higher proportion of C15–C30 materials than LSD. Increased liver weight was observed in animal treated with Bio-C, Bio-S and LSD and decreased thymus weight was found in those treated with Bio-S. Histopathological changes typical of male-rat specific hyaline-droplet nephropathy were detected in kidney tubules of animals treated with LSD, Bio-S and Bio-C. Mild adaptive changes were observed in thyroids of animals treated with LSD, Bio-S and Bio-F. Clinical chemical and biochemical changes were confined to Bio-S and LSD treated rats and included elevation in some hepatic phase-I and phase-II drug metabolizing enzymes and hepatic palmitoyl Co-A oxidase, and elevated urinary concentrations of ascorbic acid and albumin. At the given dose level of 500 mg/kg bw/day, the overall treatment-related effects of biodiesels and LSD are mild, and the severity of the treatment effects may be ranked as: LSD > Bio-S > Bio-C > Bio-F. Considered together with the presence of a higher level of water extractable materials, Bio-S may be more of a concern for potential human health than Bio-C and Bio-F in an oral exposure scenario. Further studies are needed to identify and characterize the constituents contributing to the treatment-related effects specific to these experimental biodiesels.