Two-tone pitch-comparison tasks typically comprise several successive pairs of successive tones separated by silent intervals. The serial occurrence of such pairs has been associated with degraded task performance, but the nature of this association is not fully understood. Human adult participants were presented with successive pairs of successive tones. The latter, to-be-compared tone of a pair could differ from the former, to-be-remembered tone of 1046.5 Hz by no more than ±15 Hz (25 cents). The direction of this difference was easier to identify when it was opposite to that of the preceding pair than when being the same. Merely responding accordingly (irrespectively of whether the response was correct or not) was found not to account for this finding. Our study demonstrates proactive interference in a two-tone pitch comparison task as the difficulty to remember when the first tone of the present pair occurred relative to the last tone of the immediately preceding pair.