Mutations beneficial in one environment may cause costs in different environments, resulting in antagonistic pleiotropy. Here, we describe a novel form of antagonistic pleiotropy that operates even within the same environment, where benefits and deleterious effects exhibit themselves at different growth rates. The fitness of hfq mutations in Escherichia coli affecting the RNA chaperone involved in small-RNA regulation is remarkably sensitive to growth rate. E. coli populations evolving in chemostats under nutrient limitation acquired beneficial mutations in hfq during slow growth (0.1 h(-1)) but not in populations growing sixfold faster. Four identified hfq alleles from parallel populations were beneficial at 0.1 h(-1) and deleterious at 0.6 h(-1). The hfq mutations were beneficial, deleterious or neutral at an intermediate growth rate (0.5 h(-1)) and one changed from beneficial to deleterious within a 36 min difference in doubling time. The benefit of hfq mutations was due to the greater transport of limiting nutrient, which diminished at higher growth rates. The deleterious effects of hfq mutations at 0.6 h(-1) were less clear, with decreased viability a contributing factor. The results demonstrate distinct pleiotropy characteristics in the alleles of the same gene, probably because the altered residues in Hfq affected the regulation of expression of different genes in distinct ways. In addition, these results point to a source of variation in experimental measurement of the selective advantage of a mutation; estimates of fitness need to consider variation in growth rate impacting on the magnitude of the benefit of mutations and on their fitness distributions.