Objective Mineral trioxide aggregate (MTA) mixed with water has a short working time, delayed setting, and poor consistency. A preliminary study suggested that substituting KY Jelly as a mixing vehicle improves the working properties of MTA. The present study compared the biocompatibility of white MTA mixed with water or with KY Jelly to that of Fuji II cement and of silver amalgam. Study design Periodontal ligament (PDL) cells were cultured using standard laboratory procedures. Cells were plated in wells at a density of 10,000 cells/well. The test materials were mixed and eluate placed in contact with the PDL cells. Cell viability was determined by measuring mitochondrial enzyme activity using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenytl)-2H-tetrazolium inner salt assay. Cytotoxicity was also measured in terms of cell lysis using the lactate dehydrogenase assay. The assays were completed in triplicate after time intervals of 24, 48, and 72 h. Mean cell counts were calculated and converted to a percentage of control results. A 2-way analysis of variance and Tukey honestly significant difference post-hoc test were performed to determine statistical significance. Results Eluate extractions from all materials caused significantly less cell viability and more cell death than control eluate (medium only) at all eluate time points tested. However, at 72 h the MTA/water, MTA/KY, and amalgam eluate extractions led to significantly better cell viability than the Fuji II eluates. However, there was significantly greater cell lysis for all eluates from the tested materials at 72 h than at 24 h. Conclusions Within the limitations of this in vitro study, we conclude that MTA/KY, MTA/water, and amalgam have similar biocompatibility regarding effects of their eluates on human PDL cells, and eluates from all 3 materials demonstrate better biocompatibility than eluates derived from a resin-modified glass ionomer cement.