By bioactivity-directed fractionation, three new antimicrobial and cytotoxic 5,6-dihydro-alpha-pyrones, pectinolides A-C, have been isolated from Hyptis pectinata (Lamiaceae). The absolute stereochemistry of pectinolide A [1] was established as 6S-[(3S-acetyloxy)-1Z-heptenyl]-5S-(acetyloxy)-5 ,6-dihydro-2H-pyran-2-one, on the basis of spectral, chiroptical, and chemical evidence. The structures of pectinolides B [2] and C [3] were determined as the monodeacetylated forms of 1 by comparison of their spectral data and chemical correlation with the prototype compound. Staphylococcus aureus and Bacillus subtilis were sensitive to pectinolide A [1] in the concentration range of 6.25-12.5 micrograms/ml. Compounds 1-3 exhibited significant cytotoxic activity (ED50 < 4 micrograms/ml) against a variety of tumor cell lines.