Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.