Single-molecule spectroscopy of the prototypical dye, DiI, has been investigated in rigorously de-oxygenated polymer films. The extraordinarily high signal-to-noise data demonstrate that the intrinsic T-1 --> S-0 intersystem crossing kinetics of DiI are simple-first-order with little temporal or molecule-to-molecule fluctuations. The results verify that O-2 collisions are the major factor responsible for fluctuations of the intersystem crossing rate in non-degassed and partially degassed polymer films. In addition to the intrinsic DiI intersystem kinetics, rare fluctuations are observed in the intersystem crossing rate, which are ascribed to residual impurities and/or to low quantum yield photochemical processes of DiI.