Multidrug resistance driven by ABC membrane transporters is one of the major reasons for treatment failure in human malignancy. Some limited evidence has previously been reported on the cell cycle dependence of ABC transporter expression. However, it has never been demonstrated that the functional activity of these transporters correlates with the cell cycle position. Here, we studied the rate of intrinsic ABC transport in different phases of the cell cycle in cultured MCF-7 breast cancer cells. The rate was characterized in terms of the efflux kinetics from cells loaded with an ABC transporter substrate. As averaging the kinetics over a cell population could lead to errors, we studied kinetics of ABC transport at the single-cell level. We found that the rate of ABC transport in MCF-7 cells could be described by Michaelis-Menten kinetics with two classical parameters,
V
max
and
K
M
. Each of these parameters showed similar unimodal distributions with different positions of maxima for cell subpopulations in the 2c and 4c states. Compared to the 2c cells, the 4c cells exhibited greater
V
max
values, indicating a higher
activity
of transport. They also exhibited a greater
V
max
/
K
M
ratio, indicating a higher
efficiency
of transport. Our findings suggest that cell cycle-related modulation of MDR may need to be taken into account when designing chemotherapy regimens which include cytostatic agents.