A gene which carries a bona fide loss-of-function mutation effectively becomes a functionless pseudogene, free from selective constraint. However, there is a number of molecular mechanisms that may lead to at least a partial preservation of the function of genes carrying even drastic alleles. We performed a direct measurement of the strength of negative selection acting on nonsense alleles of protein-coding genes in the Zambian population of Drosophilamelanogaster.Within those exons that carry nonsensemutations, negative selection, assayed by the ratio ofmissense over synonymous nucleotide diversity levels, appears to be absent, consistent with total loss of function. In other exons of nonsense alleles, negative selection was deeply relaxed but likely not completely absent, and the per site number of missense alleles declined significantly with the distance from the premature stop codon. This pattern may be due to alternative splicing which preserves function of some isoforms of nonsense alleles of genes.