Cultivars of Cucurbita pepo were analyzed for their isozyme phenotypes by horizontal starch gel electrophoresis. Considerable allozymic variation was observed between cultivars, especially in the aspartate aminotransferase and malate dehydrogenase isozyme systems. Each of the five fruit types represented in the 21 cultivars tested (zucchini, pumpkin, spaghetti, acorn, scallop and yellow straightneck) could be distinguished by specific allozymes or combination of allozymes. Cultivars within a fruit type gave very similar allozyme phenotypes and often could not be distinguished on the basis of the 6 assays used. Despite the outcrossing nature of the species, allozyme polymorphism within most cultivars was low and did not seriously interfere with the analysis. Approximately half of the hybrid lines tested gave heterozygous phenotypes at one or more loci, indicating that such loci could be used to screen for the percentage of hybrid seed obtained from crosses.