The photophysical properties of 1-ethyl-6-fluoro-7-(1-piperazinyl)-1,4-dihydro-4-oxoquinoline-3-carboxyl ic acid (norfloxacin, NFX) and some of its derivatives have been studied to evaluate the role of the free carboxylic acid and the nonprotonated piperazinyl group in the behavior of the 1,4-dihydro-4-oxoquinoline ring. Steady state and time-resolved fluorescence measurements at different pHs provide clear evidence in favor of singlet excited-state deactivation of NFX and its N(4')-methyl derivative pefloxacin (PFX) via intramolecular electron transfer from the N(4') atom of the piperazinyl ring to the fluoroquinolone (FQ) main system. This is a very efficient, energy-wasting pathway, which becomes dramatically enhanced in basic media. Acetylation at N(4') (as in ANFX) decreases the availability of the lone pair, making observable its fluorescence and the transient absorption spectrum of its triplet excited state even at high pH. It also reveals that the geometry of FQs changes from an almost sp(3) hybridization of the N(1') of the piperazinyl substituent in the ground state to nearly sp(2) in the singlet excited state (rehybridization accompanied by intramolecular charge transfer, RICT); accordingly, the singlet energy of ANFX is significantly lower than that of NFX and PFX The fluorescence measurements using acetonitrile as a polar nonprotic organic solvent further support deactivation of the singlet excited state of nonacetylated NFX derivatives via intramolecular electron transfer from the N(4') atom.