A series of uniflagellar mutants isolated following mutagenesis of Chlamydomonas reinhardtii (strain 137c) with ICR-191 show a remarkable positional phenotype. The flagellum that fails to develop is cis to the eyespot in more than 95% of the cells examined. Both the positional and the uniflagellar phenotypes are transmitted stably through mitotic and meiotic divisions, and in backcrosses the meiotic segregation is two mutant to two wild-type progeny. Four of the mutants, uni1, uni2, uni3 and uni4, have been studied extensively. They appear to be alleles of a single gene locus or to be closely linked (≤0.06 map units). The characteristic expression of the uniflagellar defect in cells under different growth conditions or in stable diploids indicates that the mutations alter the rate of development of the flagellum in the cis-eyespot flagellum. Electron microscopic studies suggest that the developmental defect resides in the basal body. Extensive recombination analysis to 33 nuclear markers representing the 16 linkage groups failed to establish linkage. The uni mutants, however, showed linkage to four unmapped mutant loci. Mutations for each of these loci also affect flagellar assembly.