First described in 1942,[3] FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group (−N=C=S), replacing a hydrogen atom on the bottom ring of the structure.

FITC is reactive towards nucleophiles including amine and sulfhydryl groups on proteins.

[4] A succinimidyl-ester functional group attached to the fluorescein core, creating "NHS-fluorescein", forms another common amine reactive derivative that has much greater specificity toward primary amines in the presence of other nucleophiles.

Due to the problem of photobleaching, derivatives of fluorescein such as Alexa 488 and DyLight 488 have been tailored for various chemical and biological applications where greater photostability, higher fluorescence intensity, or different attachment groups are needed.

In addition, some experiments make use of FITC's propensity for photobleaching in order to measure proteins' lateral mobility in membranes, through the technique of fluorescence recovery after photobleaching.