The expression of neutrophil-specific chemokines is known to be regulated via adenine-uridine-rich sequence elements in the 3'-untranslated regions of their mRNAs that confer a high degree of mRNA instability. Although the presence of intron sequences in eukaryotic genes is known to enhance expression, the effect of intron content on the rate of mature, translatable mRNA degradation has not been demonstrated. In this study, we have determined the effects of intron content on the rate of decay of the chemokine CXCL1 (KC) mRNA. The half-life of KC mRNA was markedly prolonged when the primary transcript was obtained from a genomic clone containing three introns as compared with the half-life observed with sequence-identical KC mRNA derived from an intron-free cDNA construct. The effect of intron content was achieved with a single intron, and neither the intron sequences nor the intron positions were critical determinants of the outcome. The intron content produced the same effect when expressed in multiple cell types and when the sequences were stably integrated into the genome. The differential decay rates were not a consequence of differential nuclear to cytoplasmic transport. The intron content of the primary transcript did not influence the rate of KC mRNA translation and did not modulate the ability of interleukin-1 stimulation to stabilize the otherwise unstable mRNA. The intron effect on mRNA decay was seen with mRNAs containing two distinct instability determinants. These findings document that intron content marks the mRNA sequence leading to enhanced stability that is particularly evident in short lived ARE-containing mRNAs.