In the present study we describe patients with non-palpable breast lesions, in which an Iodine-125 ( 125 I)-marker (or “seed”) for excision of the primary tumour and Technetium-99m nanocolloid ( 99m Tc-nanocolloid) for sentinel node biopsy (SNB) are used simultaneously. The purpose was to investigate any interference between 125 I-seeds and 99m Tc-nanocolloid by an in vitro and in vivo analysis.Contrast/interference-ratios between 125 I and 99m Tc count-rates were determined in vitro using a realistic simulation model. Measurements were performed with 3 gamma-probes with different crystal materials. In 25 consecutive patients 99m Tc-nanocolloid was intratumourally administered at the site of a previously implanted 125 I-seed. Respectively, the 125 I-setting and 99m Tc-setting of the gamma-probe guided the wide local excision and SNB and maximum counts-per-second (cps) were measured.In vitro the different probes varied in 125 I- and 99m Tc-sensitivity. The contrast-ratio between 125 I and 99m Tc in the 125 I-channel was 4.6 for a 3-month-old 125 I-seed using the most appropriate gamma-probe. In vivo the gamma-probe in the 125 I-setting measured a median of 16,300 cps at the tumour site compared to 4820 cps using the 99m Tc-setting. The 125 I-seed could be well distinguished from the 99m Tc-nanocolloid in 92% of the patients and 96% required a single operation. The SNB was successful in all patients.Simultaneous use of 125 I-seeds and 99m Tc-nanocolloid is possible under well-standardised conditions. Non-palpable breast lesions can be safely excised using the 125 I-seed in combination with a SN procedure. Use of 125 I-seeds is a next step within fine-tuning breast-conserving surgery that should lead to further investigation to confirm its value.