This paper discusses the effect of sample volume on the amount of analyte extracted from a sample by solid-phase microextraction (SPME) in two-phase (sample–fiber coating) and three-phase (sample–headspace–fiber coating) systems. Up-to-date knowledge is summarized, and new concepts are introduced. The effect of sample volume on quantification and precision of results can be neglected only in rare cases. The minimum sample volume which ensures that the amount extracted, n, is lower than 1% of the initial amount of the analyte present in the sample, as well as the volume for which exactly half of the initial amount of the analyte is extracted, have been calculated for both two- and three-phase systems. It is critical that the volumes of samples and standards are the same during analysis by SPME. Extraction kinetics in headspace analysis is dependent on the headspace capacity. If it is sufficiently large, the analyte is extracted almost exclusively from the gaseous phase, and equilibration can be very fast. On the other hand, this causes a significant loss of sensitivity. The effect of sample volume on the determination of the value of the partition coefficient, K, is also discussed. If the change in concentration of the analyte in the sample at equilibrium is not taken into account, erroneous results are obtained. Even when a proper procedure is used, there are practical limitations to the accuracy of the K value determination. Large sample volumes should always be used for K value determination, as they enable broader ranges of K values to be covered with good accuracy.