Born at Kennington Park, south London, he attended Stockwell grammar school and then King's College, then spent four years studying painting at the Royal Academy. In connection with his study he traveled to Europe and North Africa, visiting the little-known interior of Tunisia in 1879 and 1880.
In 1882 he visited southern Angola with the Earl of Mayo, and in the following year met Henry Morton Stanley in the Congo, becoming one of the first Europeans after Stanley to see the river above the Stanley Pool. His developing reputation led the Royal Geographical Society and the British Association to appoint him leader of an 1884 scientific expedition to Mount Kilimanjaro. On this expedition he concluded treaties with local chiefs (which were then transferred to the British East Africa Company), in competition German efforts to do likewise.
In October 1886 the British government appointed him vice-consul in Cameroon and the Niger River delta area, where a protectorate had been declared in 1885, and he became acting consul in 1887, deposing and banishing the local chief Ja-Ja. On leave in England in 1888, he met with Lord Salisbury and apparently helped formulate the Cape-to-Cairo plan to acquire a continuous band of territory down Africa, which he then leaked (with Salisbury's approval) to the Times in an anonymous article "by an African Explorer".
In 1889 Johnston was sent to Lisbon to negotiate the Portuguese and British spheres of influence in southeastern Africa, then went to Mozambique as consul. From there he went to Lake Nyasa to resolve the war between Arab slave-traders and the African Lakes Trading Company. Alarm over the presence of the Portuguese Serpa Pinto triggered the Anglo-Portuguese Crisis, which ended with Johnston having declared a protectorate.
This officially became the British Central Africa Protectorate in 1891, with Johnston as its first commissioner. In 1896 he was made a KCB, but afflicted by tropical fevers, transferred to Tunis as consul-general.
In 1899 Sir Harry was sent to Uganda as special commissioner to end an ongoing war. He improved the colonial administration, and in 1900 concluded the Buganda Agreement dividing the land between the UK and the chiefs.
Harry Johnston was the very model of the multi-talented African explorer; he exhibited paintings, collected flora and fauna (he was instrumental in bringing the okapi to the attention of science), climbed mountains, wrote books, signed treaties, and ruled colonial governments. He was considered unusually favorable towards the native peoples, and ran afoul of Cecil Rhodes as a result.
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