In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes, designated PYC1 and PYC2. In the wild type yeast, the expression of both genes is influenced by both the growth phase and the type of carbon source, indicating discrete regulatory mechanisms and metabolic roles for PYC1 and PYC2. On glucose minimal medium PYC1 and PYC2 are differentially regulated as shown by a constant level of PYC1 expression throughout the main growth phase compared to a high level of PYC2 expression only in the early growth phase. On ethanol minimal medium, the growth-related pattern of PYC1 and PYC2 expression was similar as shown by a 3.6-fold decline from early to mid log phase. PYC1 expression, however, was activated 10-fold above PYC2 mRNA levels during this period of growth. To further investigate the roles of the two PYC genes we determined the growth phenotypes and expression levels of PYC in pyc1 and pyc2 single null mutants. During fermentative growth, the lack of either PYC gene had little effect on the level and pattern of expression of the other PYC gene, indicating further their separate regulation. In comparison to the pyc2 null, the pyc1 null strain showed a 3- to 4-fold lower level of Pyc activity and Pyc protein concentration. Moreover, the pyc1 null showed a strong requirement for L-aspartate for efficient growth, indicating the importance of PYC1 expression for the synthesis of C4 intermediates. DV6.2 (PYC1, pyc2 delta) showed a 3.2-fold higher level of activity on ethanol minimal medium when compared to growth on glucose minimal medium, and supported growth in the absence of L-aspartate. The pyc1 null, MW21.3 (pyc1 delta, PYC2), on the other hand, did not support growth on ethanol in the absence of aspartate. This study represents the first report on the characterisation of expression of the PYC genes in yeast throughout growth. Their metabolic roles for both fermentative and gluconeogenic growth are considered.