Premature harvest of acorn squash is a widespread problem because fruits reach maximum size and optimum color within 20 days after pollination (DAP), well before peak dry matter and sugar content occur. The present study was conducted to determine the relationship between harvest date and physiological factors affecting eating quality in Cucurbita pepo L. squash. In the summer of 2005, C. pepo squash cultivars were evaluated at three harvest dates, 25, 35, and 45 days after pollination (DAP), with or without a 10-day storage period at 21 °C. Four F1 hybrid cultivars carrying powdery mildew tolerance (PMT) were evaluated: a semi-bush, commercial acorn cultivar (`Tip Top'), a high quality experimental acorn, bush hybrid (NH1634), and two sweet dumpling-type, semi-bush hybrids (NH1635 and 1636). Data were collected on mesocarp DW, oBrix (soluble solids), and partitioning of biomass between mesocarp tissue and developing embryos during storage. Peak DWs of 20% to 21% occurred at 25 DAP in NH1634, 1635 and 1636, and at 35 DAP in Tip Top (19.5 %). At 25 DAP, Brix was low (means of 5.9 to 7.2) across all cultivars. With harvest at 25 DAP plus 10 days storage, oBrix was low in Tip Top (7.1), but was higher than 10 in NH1634 and NH1636. Brix reached near maximum (13 to 15) at 45 DAP in NH1634, 1635 and 1636, and at 55 DAP in Tip Top (12). Embryos were small (DW = 8 to 19 mg) at 25 DAP and grew fairly linearly to a maximum at 55 DAP. Mean embryo DW at 55 DAP was 87.5 mg for Tip Top, 76.9 mg for NH1636, 57.1 mg for NH1634, and 28.5 mg for NH1635. The proportion of total fruit biomass expressed as energy equivalents (kJoules) allocated to embryos in mature fruit (45 DAP + 10 days storage) was 11.8% in NH1635, 18.7% in NH1634, 27.4% in Tip Top, and 30.2% in NH1636. Reallocation of assimilates from mesocarp tissue to developing embryos was a major contributing factor, along with respiration, to a reduction in mesocarp dry matter during storage.