An immunoassay demonstrated that the assembled K99 pilus on the surface of Escherichia coli grown in minimal medium appeared during the logarithmic phase of growth, but the synthesis of K99 subunits, as measured by nonequilibrium two-dimensional gel electrophoresis, occurred throughout the life cycle of the cell. Contrary to other reports, the addition of glucose to the growth medium did not affect K99 pilus assembly or subunit synthesis, although in a K99+ adenyl cyclase (cya) mutant, subunit synthesis was reduced. There was no reduction in the amount of assembled K99 on the cell surface of the cya mutant compared with the wild-type parent. The addition of L-alanine to minimal medium repressed K99 synthesis. However, if L-threonine or L-isoleucine was also included in the growth medium, the effect of L-alanine was reduced. Chloramphenicol caused a complete inhibition of K99 subunit synthesis, but assembly proceeded normally. Growth at 18° C inhibited both subunit synthesis and pilus assembly. Approximately 92% of all cellular K99 was associated with the outer membrane, and 4% was associated with the inner membrane. No K99 was detected in the cytoplasm.