The Escherichia coli urinary tract isolate C1212 contains two pyelonephritis-associated pili (pap) DNA sequences designated here as pap-17 and pap-21. Each of these pap sequences encodes antigenically-distinct pilin monomers, pilin-17 and pilin-21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin-21. Only a small fraction (5%) of strain C1212 cells expressed pilin-17. Most of the latter population simultaneously expressed pilin-21, but a low percentage of cells expressed pili composed of pilin-17 alone. In contrast, almost every E. coli K-12 cell containing multicopy pap-17 expressed pilin-17 at the cell surface. These results indicated that the regulation of pilin-17 expression observed for strain C1212 was lost when pap-17 was in the multicopy state. Transfer of pap-17 to a single copy vector resulted in a pilin-17 expression frequency lower than strain C1212 (1%). Using E. coli K-12 containing single copy pap-17, we found that the frequency of pilin-17 expression increased about 15-fold when pap-21 was present in multiple copies in trans. Subcloning of pap-21 showed that a 2.2 kilobase-pair DNA sequence adjacent to, but not including, the pilin-21 structural gene was sufficient for activation of pilin-17 expression.