PCR-based gene-targeting approaches have increased the speed of gene function analyses in ascomycetous fungi, for example, in the diploid human fungal pathogen Candida albicans. Here we describe a protocol that utilizes Rapid-PCR to amplify all cassettes available with the previously reported pFA modules. With this protocol, sufficient quantities of any cassette for use in C. albicans transformation experiments can be reliably generated in 25-50 min using either of the two alternative optimized amplification conditions; cassette amplification by standard PCR methods typically takes 3-4 h and is likely to require optimization of amplification conditions for each cassette. Transformants that appear 2-4 d after transformation can be rapidly identified using Rapid-PCR on whole cells, eliminating the need for genomic DNA extraction. In total, less than a week is required for the deletion of one allele in C. albicans. Repeating this procedure will result in the generation of homozygous mutant strains.