The hit CBBC show came under fire after it featured a sketch in which Miss Nightingale - famous for her work in the Crimean War - rejects four job applications from Jamaican-born Miss Seacole, saying the work was only 'for British girls'.
Historians said the scene was 'insulting' to the nurse's memory, and a formal complaint was made by the Nightingale Society, a collection of historians who seek to promote knowledge of her contribution to nursing.
In the sketch, the actress playing Miss Nightingale said, 'The nursing corps was for British girls. You're from Jamaica' to which Seacole responded, 'Me father was from Scotland'.
Now, the BBC's Editorial Standards Committee has ruled that the two-minute, 34-second clip's 'depiction of Florence Nightingale in relation to racial issues was materially inaccurate'.
The ESC said viewers of the clip 'would be likely to receive the overall impression that Florence Nightingale had acted towards Mary Seacole in a racially discriminatory manner'.
The Committee concluded: "Given the seriousness of any imputation of racism, the relative recency of Nightingale's life...the immensity of Nightingale's contribution to modern nursing and her significant stature in modern British history, the Committee felt it was incumbent on the programme makers to ensure that there was sound evidence upon which to base any suggestion that she had acted in a racially discriminatory manner in a Learning Zone clip.
"It appeared to the Committee that an allegation of such gravity against a person such as Nightingale required compelling proof.
"In the Committee's view, the programme makers had provided no such evidence."
A BBC spokesman said: "We note and accept the findings of the ESC. The intention of this Horrible Histories sketch was never to undermine the reputation of such an important historical figure like Florence Nightingale, but to open up a discussion of some of the attitudes of the time.
"The Learning Zone has withdrawn the sketch from their website and the episode of Horrible Histories will not be repeated in its present form."
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